Antigen library immunization

ABSTRACT

This invention is directed to antigen library immunization, which provides methods for obtaining antigens having improved properties for therapeutic and other uses. The methods are useful for obtaining improved antigens that can induce an immune response against pathogens, cancer, and other conditions, as well as antigens that are effective in modulating allergy, inflammatory and autoimmune diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.60/074,294, filed Feb. 11, 1998, and U.S. Provisional Application No.60/105,509, filed Oct. 23, 1998, which applications are incorporatedherein by reference for all purposes.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention pertains to the field of methods for developingimmunogens that can induce efficient immune responses against a broadrange of antigens.

2. Background

The interactions between pathogens and hosts are results of millions ofyears of evolution, during which the mammalian immune system has evolvedsophisticated means to counterattack pathogen invasions. However,bacterial and viral pathogens have simultaneously gained a number ofmechanisms to improve their virulence and survival in hosts, providing amajor challenge for vaccine research and development despite the powersof modern techniques of molecular and cellular biology. Similar to theevolution of pathogen antigens, several cancer antigens are likely tohave gained means to downregulate their immunogenicity as a mechanism toescape the host immune system.

Efficient vaccine development is also hampered by the antigenicheterogeneity of different strains of pathogens, driven in part byevolutionary forces as means for the pathogens to escape immunedefenses. Pathogens also reduce their immunogenicity by selectingantigens that are difficult to express, process and/or transport in hostcells, thereby reducing the availability of immunogenic peptides to themolecules initiating and modulating immune responses. The mechanismsassociated with these challenges are complex, multivariate and ratherpoorly characterized. Accordingly, a need exists for vaccines that caninduce a protective immune response against bacterial and viralpathogens. The present invention fulfills this and other needs.

SUMMARY OF THE INVENTION

The present invention provides recombinant multivalent antigenicpolypeptides that include a first antigenic determinant from a firstdisease-associated polypeptide and at least a second antigenicdeterminant from a second disease-associated polypeptide. Thedisease-associated polypeptides can be selected from the groupconsisting of cancer antigens, antigens associated with autoimmunitydisorders, antigens associated with inflammatory conditions, antigensassociated with allergic reactions, antigens associated with infectiousagents, and other antigens that are associated with a disease condition.

In another embodiment, the invention provides a recombinant antigenlibrary that contains recombinant nucleic acids that encode antigenicpolypeptides. The libraries are typically obtained by recombining atleast first and second forms of a nucleic acid which includes apolynucleotide sequence that encodes a disease-associated antigenicpolypeptide, wherein the first and second forms differ from each otherin two or more nucleotides, to produce a library of recombinant nucleicacids.

Another embodiment of the invention provides methods of obtaining apolynucleotide that encodes a recombinant antigen having improvedability to induce an immune response to a disease condition. Thesemethods involve: (1) recombining at least first and second forms of anucleic acid which comprises a polynucleotide sequence that encodes anantigenic polypeptide that is associated with the disease condition,wherein the first and second forms differ from each other in two or morenucleotides, to produce a library of recombinant nucleic acids; and (2)screening the library to identify at least one optimized recombinantnucleic acid that encodes an optimized recombinant antigenic polypeptidethat has improved ability to induce an immune response to the diseasecondition.

These methods optionally further involve: (3) recombining at least oneoptimized recombinant nucleic acid with a further form of the nucleicacid, which is the same or different from the first and second forms, toproduce a farther library of recombinant nucleic acids; (4) screeningthe further library to identify at least one further optimizedrecombinant nucleic acid that encodes a polypeptide that has improvedability to induce an immune response to the disease condition; and (5)repeating (3) and (4), as necessary, until the further optimizedrecombinant nucleic acid encodes a polypeptide that has improved abilityto induce an immune response to the disease condition.

In some embodiments, the optimized recombinant nucleic acid encodes amultivalent antigenic polypeptide and the screening is accomplished byexpressing the library of recombinant nucleic acids in a phage displayexpression vector such that the recombinant antigen is expressed as afusion protein with a phage polypeptide that is displayed on a phageparticle surface; contacting the phage with a first antibody that isspecific for a first serotype of the pathogenic agent and selectingthose phage that bind to the first antibody; and contacting those phagethat bind to the first antibody with a second antibody that is specificfor a second serotype of the pathogenic agent and selecting those phagethat bind to the second antibody; wherein those phage that bind to thefirst antibody and the second antibody express a multivalent antigenicpolypeptide.

The invention also provides methods of obtaining a recombinant viralvector which has an enhanced ability to induce an antiviral response ina cell. These methods can include the steps of: (1) recombining at leastfirst and second forms of a nucleic acid which comprise a viral vector,wherein the first and second forms differ from each other in two or morenucleotides, to produce a library of recombinant viral vectors; (2)transfecting the library of recombinant viral vectors into a populationof mammalian cells; (3) staining the cells for the presence of Mxprotein; and (4) isolating recombinant viral vectors from cells whichstain positive for Mx protein, wherein recombinant viral vectors frompositive staining cells exhibit enhanced ability to induce an antiviralresponse.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic representation of a method for generating achimeric, multivalent antigen that has immunogenic regions from multipleantigens. Antibodies to each of the non-chimeric parental immunogenicpolypeptides are specific for the respective organisms (A, B, C). Aftercarrying out the recombination and selection methods of the invention,however, a chimeric immunogenic polypeptide is obtained that isrecognized by antibodies raised against each of the three parentalimmunogenic polypeptides.

FIG. 2 shows the principle of family DNA shuffling. A family of antigengenes from related pathogens are subjected to shuffling, which resultsin a library of chimeric and/or mutated antigens. Screening methods areemployed to identify those recombinant antigens that are the mostimmunogenic and/or cross-protective. These can, if desired, be subjectedto additional rounds of shuffling and screening.

FIG. 3A-FIG. 3B shows a schematic for a method by which one can obtainrecombinant polypeptides that can induce a broad-spectrum immuneresponse. In FIG. 3A, wild-type immunogenic polypeptides from thepathogens A, B, and C provide protection against the correspondingpathogen from which the polypeptide is derived, but little or nocross-protection against the other pathogens (left panel). Aftershuffling, an A/B/C chimeric polypeptide is obtained that can induce aprotective immune response against all three pathogen types (rightpanel). In FIG. 3B, shuffling is used with substrate nucleic acids fromtwo pathogen strains (A, B), which encode polypeptides that areprotective only against the corresponding pathogen. After shuffling, theresulting chimeric polypeptide can induce an immune response that iseffective against not only the two parental pathogen strains, but alsoagainst a third strain of pathogen (C).

FIG. 4 diagrams some of the possible factors that can determine whethera particular polynucleotide encodes an immunogenic polypeptide having adesired property, such as enhanced immunogenicity and/orcross-reactivity. Those sequence regions that positively affect aparticular property are indicated as plus signs along the antigen gene,while those sequence regions that have a negative effect are shown asminus signs. A pool of related antigen genes are shuffled and screenedto obtain those that recombinant nucleic acids that have gained positivesequence regions and lost negative regions. No pre-existing knowledge asto which regions are positive or negative for a particular trait isrequired.

FIG. 5 shows a schematic representation of the screening strategy forantigen library screening.

FIG. 6 shows a schematic representation of a strategy for pooling anddeconvolution as used in antigen library screening.

FIG. 7 is an alignment of the nucleotide sequences of glycoprotein D(gD) from HSV-1 (SEQ ID NO: 1) and HSV-2 (gD-1 (SEQ ID NO: 2) and gD-2(SEQ ID NO: 3)).

FIG. 8A shows a diagram of a method for expressing HIV gp120 usinggenetic vaccine vectors and generation of a library of shuffled gp120genes. FIG. 8B shows PCR primers that are useful for obtaining gp120nucleic acid substrates for DNA shuffling reactions. Primers suitablefor generating substrates include 6025F (SEQ ID NO: 4), 7773R (SEQ IDNO: 5), and primers suitable for amplifying the shuffled nucleic acidsinclude 6196F (SEQ ID NO: 6) and 7746R (SEQ ID NO: 7). The primerBssH2-6205F (SEQ ID NO: 8) can be used to clone the resulting fragmentinto a genetic vaccine vector.

FIG. 9 shows the domain structure of hepadnavirus envelope genes.

FIG. 10 shows a schematic representation of the use of shuffling toobtain hepadnavirus proteins in which the immunogenicity of oneantigenic domain is improved.

FIG. 11 shows a strategy in which genes that encode the hepadnavirusproteins having one antigenic domain that has improved immunogenicityare shuffled to obtain recombinant proteins in which all three domainshave improved immunogenicity.

FIG. 12 shows the transmembrane organization of the HBsAg polypeptide.

FIG. 13 shows a method for using phage display to obtain recombinantallergens that are not bound by pre-existing IgE.

FIG. 14 shows a strategy for screening of recombinant allergens toidentify those that are effective in activating T_(H) cells. PBMC or Tcell clones from atopic individuals are exposed to antigen-presentingcells that display the antigen variants obtained using the methods ofthe invention. To identify those allergen variants that are effective inactivating T cells, the cultures are tested for induction of T cellproliferation or for a pattern of cytokine synthesis that is indicativeof the particular type of T cell activation that is desired. If desired,the allergen variants that test positive in the in vitro assay can besubjected to in vivo testing.

FIG. 15 shows a strategy for screening of recombinant cancer antigens toidentify those that are effective in activating T cells of cancerpatients.

FIG. 16A and FIG. 16B show two different strategies for generatingvectors that contain multiple T cell epitopes obtained, for example, byDNA shuffling. In FIG. 16A, each individual shuffled epitope-encodingnucleic acid is linked to a single promoter, and multiplepromoter-epitope gene constructs can be placed in a single vector. Thescheme shown in FIG. 16B involves linking multiple epitope-encodingnucleic acids to a single promoter.

FIG. 17 shows the sequences of PreS2-S coding regions and correspondingamino acid sequences of different hepatitis B surface antigen (HBsAg) orwoodchuck hepatitis B (WHV) proteins. Primers suitable for amplificationof this region are also shown.

FIG. 18 shows primers that are suitable for amplification of largefragments that contain the S2S coding sequences. The primers hybridizeto regions that are approximately 200 bp outside the desired sequences.

FIG. 19 shows an alignment of the amino acid sequences of surfaceantigens from different HVB and WHV subtypes.

FIG. 20 shows a diagram of multimeric particles that assemble when anappropriate number of chimeric polypeptides and native HBsAg S monomersare mixed.

DETAILED DESCRIPTION

Definitions

The term “screening” describes, in general, a process that identifiesoptimal antigens. Several properties of the antigen can be used inselection and screening including antigen expression, folding,stability, immunogenicity and presence of epitopes from several relatedantigens. Selection is a form of screening in which identification andphysical separation are achieved simultaneously by expression of aselection marker, which, in some genetic circumstances, allows cellsexpressing the marker to survive while other cells die (or vice versa).Screening markers include, for example, luciferase, beta-galactosidaseand green fluorescent protein. Selection markers include drug and toxinresistance genes, and the like. Because of limitations in studyingprimary immune responses in vitro, in vivo studies are particularlyuseful screening methods. In these studies, the antigens are firstintroduced to test animals, and the immune responses are subsequentlystudied by analyzing protective immune responses or by studying thequality or strength of the induced immune response using lymphoid cellsderived from the immunized animal. Although spontaneous selection canand does occur in the course of natural evolution, in the presentmethods selection is performed by man.

A “exogenous DNA segment”, “heterologous sequence” or a “heterologousnucleic acid”, as used herein, is one that originates from a sourceforeign to the particular host cell, or, if from the same source, ismodified from its original form. Thus, a heterologous gene in a hostcell includes a gene that is endogenous to the particular host cell, buthas been modified. Modification of a heterologous sequence in theapplications described herein typically occurs through the use of DNAshuffling. Thus, the terms refer to a DNA segment which is foreign orheterologous to the cell, or homologous to the cell but in a positionwithin the host cell nucleic acid in which the element is not ordinarilyfound. Exogenous DNA segments are expressed to yield exogenouspolypeptides.

The term “gene” is used broadly to refer to any segment of DNAassociated with a biological function. Thus, genes include codingsequences and/or the regulatory sequences required for their expression.Genes also include nonexpressed DNA segments that, for example, formrecognition sequences for other proteins. Genes can be obtained from avariety of sources, including cloning from a source of interest orsynthesizing from known or predicted sequence information, and mayinclude sequences designed to have desired parameters.

The term “isolated”, when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is essentially free of other cellularcomponents with which it is associated in the natural state. It ispreferably in a homogeneous state although it can be in either a dry oraqueous solution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinwhich is the predominant species present in a preparation issubstantially purified. In particular, an isolated gene is separatedfrom open reading frames which flank the gene and encode a protein otherthan the gene of interest. The term “purified” denotes that a nucleicacid or protein gives rise to essentially one band in an electrophoreticgel. Particularly, it means that the nucleic acid or protein is at leastabout 50% pure, more preferably at least about 85% pure, and mostpreferably at least about 99% pure.

The term “naturally-occurring” is used to describe an object that can befound in nature as distinct from being artificially produced by man. Forexample, a polypeptide or polynucleotide sequence that is present in anorganism (including viruses, bacteria, protozoa, insects, plants ormammalian tissue) that can be isolated from a source in nature and whichhas not been intentionally modified by man in the laboratory isnaturally-occurring.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form. Unless specifically limited, the term encompassesnucleic acids containing known analogues of natural nucleotides whichhave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.degenerate codon substitutions) and complementary sequences and as wellas the sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al. (1991) NucleicAcid Res. 19: 5081; Ohtsuka et al. (1985) J. Biol. Chem. 260: 2605-2608;Cassol et al. (1992); Rossolini et al. (1994) Mol. Cell. Probes 8:91-98). The term nucleic acid is used interchangeably with gene, cDNA,and mRNA encoded by a gene.

“Nucleic acid derived from a gene” refers to a nucleic acid for whosesynthesis the gene, or a subsequence thereof, has ultimately served as atemplate. Thus, an mRNA, a cDNA reverse transcribed from an mRNA, an RNAtranscribed from that cDNA, a DNA amplified from the cDNA, an RNAtranscribed from the amplified DNA, etc., are all derived from the geneand detection of such derived products is indicative of the presenceand/or abundance of the original gene and/or gene transcript in asample.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itincreases the transcription of the coding sequence. Operably linkedmeans that the DNA sequences being linked are typically contiguous and,where necessary to join two protein coding regions, contiguous and inreading frame. However, since enhancers generally function whenseparated from the promoter by several kilobases and intronic sequencesmay be of variable lengths, some polynucleotide elements may be operablylinked but not contiguous.

A specific binding affinity between two molecules, for example, a ligandand a receptor, means a preferential binding of one molecule for anotherin a mixture of molecules. The binding of the molecules can beconsidered specific if the binding affinity is about 1×10⁴ M⁻¹ to about1×10⁶ M⁻¹ or greater.

The term “recombinant” when used with reference to a cell indicates thatthe cell replicates a heterologous nucleic acid, or expresses a peptideor protein encoded by a heterologous nucleic acid. Recombinant cells cancontain genes that are not found within the native (non-recombinant)form of the cell. Recombinant cells can also contain genes found in thenative form of the cell wherein the genes are modified and re-introducedinto the cell by artificial means. The term also encompasses cells thatcontain a nucleic acid endogenous to the cell that has been modifiedwithout removing the nucleic acid from the cell; such modificationsinclude those obtained by gene replacement, site-specific mutation, andrelated techniques.

A “recombinant expression cassette” or simply an “expression cassette”is a nucleic acid construct, generated recombinantly or synthetically,with nucleic acid elements that are capable of effecting expression of astructural gene in hosts compatible with such sequences. Expressioncassettes include at least promoters and optionally, transcriptiontermination signals. Typically, the recombinant expression cassetteincludes a nucleic acid to be transcribed (e.g., a nucleic acid encodinga desired polypeptide), and a promoter. Additional factors necessary orhelpful in effecting expression may also be used as described herein.For example, an expression cassette can also include nucleotidesequences that encode a signal sequence that directs secretion of anexpressed protein from the host cell. Transcription termination signals,enhancers, and other nucleic acid sequences that influence geneexpression, can also be included in an expression cassette.

A “multivalent antigenic polypeptide” or a “recombinant multivalentantigenic polypeptide” is a non-naturally occurring polypeptide thatincludes amino acid sequences from more than one source polypeptide,which source polypeptide is typically a naturally occurring polypeptide.At least some of the regions of different amino acid sequencesconstitute epitopes that are recognized by antibodies found in a mammalthat has been injected with the source polypeptide. The sourcepolypeptides from which the different epitopes are derived are usuallyhomologous (i.e., have the same or a similar structure and/or function),and are often from different isolates, serotypes, strains, species, oforganism or from different disease states, for example.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acid or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the following sequence comparison algorithms or by visual inspection.

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides, refers to two or more sequences or subsequencesthat have at least 60%, preferably 80%, most preferably 90-95%nucleotide or amino acid residue identity, when compared and aligned formaximum correspondence, as measured using one of the following sequencecomparison algorithms or by visual inspection. Preferably, thesubstantial identity exists over a region of the sequences that is atleast about 50 residues in length, more preferably over a region of atleast about 100 residues, and most preferably the sequences aresubstantially identical over at least about 150 residues. In someembodiments, the sequences are substantially identical over the entirelength of the coding regions.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul (1993) Proc. Nat'l. Acad.Sci. USA 90:5873-5787). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

Another indication that two nucleic acid sequences are substantiallyidentical is that the two molecules hybridize to each other understringent conditions. The phrase “hybridizing specifically to”, refersto the binding, duplexing, or hybridizing of a molecule only to aparticular nucleotide sequence under stringent conditions when thatsequence is present in a complex mixture (e.g., total cellular) DNA orRNA. “Bind(s) substantially” refers to complementary hybridizationbetween a probe nucleic acid and a target nucleic acid and embracesminor mismatches that can be accommodated by reducing the stringency ofthe hybridization media to achieve the desired detection of the targetpolynucleotide sequence.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and northern hybridizations are sequence dependent, andare different under different environmental parameters. Longer sequenceshybridize specifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen (1993) LaboratoryTechniques in Biochemistry and Molecular Biology—Hybridization withNucleic Acid Probes part I chapter 2 “Overview of principles ofhybridization and the strategy of nucleic acid probe assays”, Elsevier,New York. Generally, highly stringent hybridization and wash conditionsare selected to be about 5° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength and pH.Typically, under “stringent conditions” a probe will hybridize to itstarget subsequence, but to no other sequences.

The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe. Very stringent conditions are selected to be equal to the T_(m)for a particular probe. An example of stringent hybridization conditionsfor hybridization of complementary nucleic acids which have more than100 complementary residues on a filter in a Southern or northern blot is50% formamide with 1 mg of heparin at 42° C., with the hybridizationbeing carried out overnight. An example of highly stringent washconditions is 0.15M NaCl at 72° C. for about 15 minutes. An example ofstringent wash conditions is a 0.2×SSC wash at 65° C. for 15 minutes(see, Sambrook, infra., for a description of SSC buffer). Often, a highstringency wash is preceded by a low stringency wash to removebackground probe signal. An example medium stringency wash for a duplexof, e.g., more than 100 nucleotides, is 1×SSC at 45° C. for 15 minutes.An example low stringency wash for a duplex of, e.g., more than 100nucleotides, is 4-6×SSC at 40° C. for 15 minutes. For short probes(e.g., about 10 to 50 nucleotides), stringent conditions typicallyinvolve salt concentrations of less than about 1.0 M Na⁺ ion, typicallyabout 0.01 to 1.0 M Na⁺ ion concentration (or other salts) at pH 7.0 to8.3, and the temperature is typically at least about 30° C. Stringentconditions can also be achieved with the addition of destabilizingagents such as formamide. In general, a signal to noise ratio of 2× (orhigher) than that observed for an unrelated probe in the particularhybridization assay indicates detection of a specific hybridization.Nucleic acids which do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, e.g., when a copyof a nucleic-acid is created using the maximum codon degeneracypermitted by the genetic code.

A further indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with, or specificallybinds to, the polypeptide encoded by the second nucleic acid. Thus, apolypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions.

The phrase “specifically (or selectively) binds to an antibody” or“specifically (or selectively) immunoreactive with”, when referring to aprotein or peptide, refers to a binding reaction which is determinativeof the presence of the protein, or an epitope from the protein, in thepresence of a heterogeneous population of proteins and other biologics.Thus, under designated immunoassay conditions, the specified antibodiesbind to a particular protein and do not bind in a significant amount toother proteins present in the sample. The antibodies raised against amultivalent antigenic polypeptide will generally bind to the proteinsfrom which one or more of the epitopes were obtained. Specific bindingto an antibody under such conditions may require an antibody that isselected for its specificity for a particular protein. A variety ofimmunoassay formats may be used to select antibodies specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays, Western blots, or immunohistochemistry are routinely usedto select monoclonal antibodies specifically immunoreactive with aprotein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual,Cold Spring Harbor Publications, New York “Harlow and Lane”), for adescription of immunoassay formats and conditions that can be used todetermine specific immunoreactivity. Typically a specific or selectivereaction will be at least twice background signal or noise and moretypically more than 10 to 100 times background.

“Conservatively modified variations” of a particular polynucleotidesequence refers to those polynucleotides that encode identical oressentially identical amino acid sequences, or where the polynucleotidedoes not encode an amino acid sequence, to essentially identicalsequences. Because of the degeneracy of the genetic code, a large numberof functionally identical nucleic acids encode any given polypeptide.For instance, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode theamino acid arginine. Thus, at every position where an arginine isspecified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of “conservatively modified variations.” Every polynucleotidesequence described herein which encodes a polypeptide also describesevery possible silent variation, except where otherwise noted. One ofskill will recognize that each codon in a nucleic acid (except AUG,which is ordinarily the only codon for methionine) can be modified toyield a functionally identical molecule by standard techniques.Accordingly, each “silent variation” of a nucleic acid which encodes apolypeptide is implicit in each described sequence.

Furthermore, one of skill will recognize that individual substitutions,deletions or additions which alter, add or delete a single amino acid ora small percentage of amino acids (typically less than 5%, moretypically less than 1%) in an encoded sequence are “conservativelymodified variations” where the alterations result in the substitution ofan amino acid with a chemically similar amino acid. Conservativesubstitution tables providing functionally similar amino acids are wellknown in the art. The following five groups each contain amino acidsthat are conservative substitutions for one another:

-   -   Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L),        Isoleucine (I);    -   Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W);    -   Sulfur-containing: Methionine (M), Cysteine (C);    -   Basic: Arginine (R), Lysine (K), Histidine (H);    -   Acidic: Aspartic acid (D), Glutamic acid (E), Asparagine (N),        Glutamine (Q). See also, Creighton (1984) Proteins, W.H. Freeman        and Company, for additional groupings of amino acids. In        addition, individual substitutions, deletions or additions which        alter, add or delete a single amino acid or a small percentage        of amino acids in an encoded sequence are also “conservatively        modified variations”.

A “subsequence” refers to a sequence of nucleic acids or amino acidsthat comprise a part of a longer sequence of nucleic acids or aminoacids (e.g., polypeptide) respectively.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention provides a new approach to vaccine development, which istermed “antigen library immunization.” No other technologies areavailable for generating libraries of related antigens or optimizingknown protective antigens. The most powerful previously existing methodsfor identification of vaccine antigens, such as high throughputsequencing or expression library immunization, only explore the sequencespace provided by the pathogen genome. These approaches are likely to beinsufficient, because they generally only target single pathogenstrains, and because natural evolution has directed pathogens todownregulate their own immunogenicity. In contrast, the immunizationprotocols of the invention, which use shuffled antigen libraries,provide a means to identify novel antigen sequences. Those antigens thatare most protective can be selected from these pools by in vivochallenge models. Antigen library immunization dramatically expands thediversity of available immunogen sequences, and therefore, these antigenchimera libraries can also provide means to defend against newlyemerging pathogen variants of the future. The methods of the inventionenable the identification of individual chimeric antigens that provideefficient protection against a variety of existing pathogens, providingimproved vaccines for troops and civilian populations.

The methods of the invention provide an evolution-based approach, suchas DNA shuffling in particular, that is an optimal approach to improvethe immunogenicity of many types of antigens. For example, the methodsprovide means of obtaining optimized cancer antigens useful forpreventing and treating malignant diseases. Furthermore, an increasingnumber of self-antigens, causing autoimmune diseases, and allergens,causing atopy, allergy and asthma, have been characterized. Theimmunogenicity and manufacturing of these antigens can likewise beimproved with the methods of this invention.

The antigen library immunization methods of the invention provide ameans by which one can obtain a recombinant antigen that has improvedability to induce an immune response to a pathogenic agent. A“pathogenic agent” refers to an organism or virus that is capable ofinfecting a host cell. Pathogenic agents typically include and/or encodea molecule, usually a polypeptide, that is immunogenic in that an immuneresponse is raised against the immunogenic polypeptide. Often, theimmune response raised against an immunogenic polypeptide from oneserotype of the pathogenic agent is not capable of recognizing, and thusprotecting against, a different serotype of the pathogenic agent, orother related pathogenic agents. In other situations, the polypeptideproduced by a pathogenic agent is not produced in sufficient amounts, oris not sufficiently immunogenic, for the infected host to raise aneffective immune response against the pathogenic agent.

These problems are overcome by the methods of the invention, whichtypically involve recombining two or more forms of a nucleic acid thatencode a polypeptide of the pathogenic agent, or antigen involved inanother disease or condition. These recombination methods, referred toherein as “DNA shuffling”, use as substrates forms of the nucleic acidthat differ from each other in two or more nucleotides, so a library ofrecombinant nucleic acids results. The library is then screened toidentify at least one optimized recombinant nucleic acid that encodes anoptimized recombinant antigen that has improved ability to induce animmune response to the pathogenic agent or other condition. Theresulting recombinant antigens often are chimeric in that they arerecognized by antibodies (Abs) reacting against multiple pathogenstrains, and generally can also elicit broad spectrum immune responses.Specific neutralizing antibodies are known to mediate protection againstseveral pathogens of interest, although additional mechanisms, such ascytotoxic T lymphocytes, are likely to be involved. The concept ofchimeric, multivalent antigens inducing broadly reacting antibodyresponses is further illustrated in FIG. 1.

In preferred embodiments, the different forms of the nucleic acids thatencode antigenic polypeptides are obtained from members of a family ofrelated pathogenic agents. This scheme of performing DNA shuffling usingnucleic acids from related organisms, known as “family shuffling,” isdescribed in Crameri et al. ((1998) Nature 391: 288-291) and is shownschematically in FIG. 2. Polypeptides of different strains and serotypesof pathogens generally vary between 60-98%, which will allow forefficient family DNA shuffling. Therefore, family DNA shuffling providesan effective approach to generate multivalent, crossprotective antigens.The methods are useful for obtaining individual chimeras thateffectively protect against most or all pathogen variants (FIG. 3A).Moreover, immunizations using entire libraries or pools of shuffledantigen chimeras can also result in identification of chimeric antigensthat protect against pathogen variants that were not included in thestarting population of antigens (for example, protection against strainC by shuffled library of chimeras/mutants of strains A and B in FIG.3B). Accordingly, the antigen library immunization approach enables thedevelopment of immunogenic polypeptides that can induce immune responsesagainst poorly characterized, newly emerging pathogen variants.

Sequence recombination can be achieved in many different formats andpermutations of formats, as described in further detail below. Theseformats share some common principles. For example, the targets formodification vary in different applications, as does the property soughtto be acquired or improved. Examples of candidate targets foracquisition of a property or improvement in a property include genesthat encode proteins which have immunogenic and/or toxigenic activitywhen introduced into a host organism.

The methods use at least two variant forms of a starting target. Thevariant forms of candidate substrates can show substantial sequence orsecondary structural similarity with each other, but they should alsodiffer in at least one and preferably at least two positions. Theinitial diversity between forms can be the result of natural variation,e.g., the different variant forms (homologs) are obtained from differentindividuals or strains of an organism, or constitute related sequencesfrom the same organism (e.g., allelic variations), or constitutehomologs from different organisms (interspecific variants).Alternatively, initial diversity can be induced, e.g., the variant formscan be generated by error-prone transcription, such as an error-pronePCR or use of a polymerase which lacks proof-reading activity (see, Liao(1990) Gene 88:107-111), of the first variant form, or, by replicationof the first form in a mutator strain (mutator host cells are discussedin further detail below, and are generally well known). A mutator straincan include any mutants in any organism impaired in the functions ofmismatch repair. These include mutant gene products of mutS, mutT, mutH,mutL, ovrD, dcm, vsr, umuC, umuD, sbcB, recJ, etc. The impairment isachieved by genetic mutation, allelic replacement, selective inhibitionby an added reagent such as a small compound or an expressed antisenseRNA, or other techniques. Impairment can be of the genes noted, or ofhomologous genes in any organism. Other methods of generating initialdiversity include methods well known to those of skill in the art,including, for example, treatment of a nucleic acid with a chemical orother mutagen, through spontaneous mutation, and by inducing anerror-prone repair system (e.g., SOS) in a cell that contains thenucleic acid. The initial diversity between substrates is greatlyaugmented in subsequent steps of recombination for library generation.

Properties Involved in Immunogenicity

The effectiveness of an antigen in inducing an immune response against apathogen can depend upon several factors, many of which are not wellunderstood. Most previously available methods for increasing theeffectiveness of antigens are dependent upon understanding the molecularbasis for these factors. However, DNA shuffling and antigen libraryimmunization according to the methods of the invention are effectiveeven where the molecular bases are unknown. The methods of the inventiondo not rely upon a priori assumptions.

Polynucleotide sequences that can positively or negatively affect theimmunogenicity of an antigen encoded by the polynucleotide are oftenscattered throughout the entire antigen gene. Several of these factorsare shown diagrammatically in FIG. 4. By recombining different forms ofpolynucleotide that encode the antigen using DNA shuffling, followed byselection for those chimeric polynucleotides that encode an antigen thatcan induce an improved immune response, one can obtain primarilysequences that have a positive influence on antigen immunogenicity.Those sequences that negatively affect antigen immunogenicity areeliminated (FIG. 4). One need not know the particular sequencesinvolved.

DNA Shuffling Methods

Generally, the methods of the invention entail performing DNArecombination (“shuffling”) and screening or selection to “evolve”individual genes, whole plasmids or viruses, multigene clusters, or evenwhole genomes (Stemmer (1995) Bio/Technology 13:549-553). Reiterativecycles of recombination and screening/selection can be performed tofurther evolve the nucleic acids of interest. Such techniques do notrequire the extensive analysis and computation required by conventionalmethods for polypeptide engineering. Shuffling allows the recombinationof large numbers of mutations in a minimum number of selection cycles,in contrast to natural pair-wise recombination events (e.g., as occurduring sexual replication). Thus, the sequence recombination techniquesdescribed herein provide particular advantages in that they providerecombination between mutations in any or all of these, therebyproviding a very fast way of exploring the manner in which differentcombinations of mutations can affect a desired result. In someinstances, however, structural and/or functional information isavailable which, although not required for sequence recombination,provides opportunities for modification of the technique.

The DNA shuffling methods of the invention can involve at least one ofat least four different approaches to improve immunogenic activity aswell as to broaden specificity. First, DNA shuffling can be performed ona single gene. Secondly, several highly homologous genes can beidentified by sequence comparison with known homologous genes. Thesegenes can be synthesized and shuffled as a family of homologs, to selectrecombinants with the desired activity. The shuffled genes can be clonedinto appropriate host cells, such as E. coli, yeast, plants, fungi,animal cells, and the like, and those that encode antigens having thedesired properties can be identified by the methods described below.Third, whole genome shuffling can be performed to shuffle genes thatencode antigenic polypeptides (along with other genomic nucleic acids).For whole genome shuffling approaches, it is not even necessary toidentify which genes are being shuffled. Instead, e.g., bacterial cellor viral genomes are combined and shuffled to acquire recombinantpolypeptides that have enhanced ability to induce an immune response, asmeasured in any of the assays described below. Fourth, antigenicpolypeptide-encoding genes can be codon modified to access mutationaldiversity not present in any naturally occurring gene. Details on eachof these procedures can be found below.

Exemplary formats and examples for sequence recombination, sometimesreferred to as DNA shuffling, evolution, or molecular breeding, havebeen described by the present inventors and co-workers in co-pendingapplications U.S. patent application Ser. No. 08/198,431, filed Feb. 17,1994, Serial No. PCT/US95/02126, filed, Feb. 17, 1995, Ser. No.08/425,684, filed Apr. 18, 1995, Ser. No. 08/537,874, filed Oct. 30,1995, Ser. No. 08/564,955, filed Nov. 30, 1995, Ser. No. 08/621,859,filed Mar. 25, 1996, Ser. No. 08/621,430, filed Mar. 25, 1996, SerialNo. PCT/US96/05480, filed Apr. 18, 1996, Ser. No. 08/650,400, filed May20, 1996, Ser. No. 08/675,502, filed Jul. 3, 1996, Ser. No. 08/721, 824,filed Sep. 27, 1996, Serial No. PCT/US97/17300, filed Sep. 26, 1997, andSerial No. PCT/US97/24239, filed Dec. 17, 1997; Stemmer, Science270:1510 (1995); Stemmer et al., Gene 164:49-53 (1995); Stemmer,Bio/Technology 13:549-553 (1995); Stemmer, Proc. Natl. Acad. Sci. U.S.A.91:10747-10751 (1994); Stemmer, Nature 370:389-391 (1994); Crameri etal., Nature Medicine 2(1):1-3 (1996); Crameri et al., NatureBiotechnology 14:315-319 (1996), each of which is incorporated byreference in its entirety for all purposes.

Other methods for obtaining recombinant polynucleotides and/or forobtaining diversity in nucleic acids used as the substrates forshuffling include, for example, homologous recombination(PCT/US98/05223; Publ. No. WO98/42727); oligonucleotide-directedmutagenesis (for review see, Smith, Ann. Rev. Genet. 19: 423-462 (1985);Botstein and Shortle, Science 229: 1193-1201 (1985); Carter, Biochem. J.237: 1-7 (1986); Kunkel, “The efficiency of oligonucleotide directedmutagenesis” in Nucleic acids & Molecular Biology, Eckstein and Lilley,eds., Springer Verlag, Berlin (1987)). Included among these methods areoligonucleotide-directed mutagenesis (Zoller and Smith, Nucl. Acids Res.10: 6487-6500 (1982), Methods in Enzymol. 100: 468-500 (1983), andMethods in Enzymol. 154: 329-350 (1987)) phosphothioate-modified DNAmutagenesis (Taylor et al., Nucl. Acids Res. 13: 8749-8764 (1985);Taylor et al., Nucl. Acids Res. 13: 8765-8787 (1985); Nakamaye andEckstein, Nucl. Acids Res. 14: 9679-9698 (1986); Sayers et al., Nucl.Acids Res. 16: 791-802 (1988); Sayers et al., Nucl. Acids Res. 16:803-814 (1988)), mutagenesis using uracil-containing templates (Kunkel,Proc. Nat'l. Acad. Sci. USA 82: 488-492 (1985) and Kunkel et al.,Methods in Enzymol. 154: 367-382)); mutagenesis using gapped duplex DNA(Kramer et al., Nucl. Acids Res. 12: 9441-9456 (1984); Kramer and Fritz,Methods in Enzymol. 154: 350-367 (1987); Kramer et al., Nucl. Acids Res.16: 7207 (1988)); and Fritz et al., Nucl. Acids Res. 16: 6987-6999(1988)). Additional suitable methods include point mismatch repair(Kramer et al., Cell 38: 879-887 (1984)), mutagenesis usingrepair-deficient host strains (Carter et al., Nucl. Acids Res. 13:4431-4443 (1985); Carter, Methods in Enzymol. 154: 382-403 (1987)),deletion mutagenesis (Eghtedarzadeh and Henikoff, Nucl. Acids Res. 14:5115 (1986)), restriction-selection and restriction-purification (Wellset al., Phil. Trans. R. Soc. Lond. A 317: 415-423 (1986)), mutagenesisby total gene synthesis (Nambiar et al., Science 223: 1299-1301 (1984);Sakamar and Khorana, Nucl. Acids Res. 14: 6361-6372 (1988); Wells etal., Gene 34: 315-323 (1985); and Grundström et al., Nucl. Acids Res.13: 3305-3316 (1985). Kits for mutagenesis are commercially available(e.g., Bio-Rad, Amersham International, Anglian Biotechnology).

The breeding procedure starts with at least two substrates thatgenerally show some degree of sequence identity to each other (i.e., atleast about 30%, 50%, 70%, 80% or 90% sequence identity), but differfrom each other at certain positions. The difference can be any type ofmutation, for example, substitutions, insertions and deletions. Often,different segments differ from each other in about 5-20 positions. Forrecombination to generate increased diversity relative to the startingmaterials, the starting materials must differ from each other in atleast two nucleotide positions. That is, if there are only twosubstrates, there should be at least two divergent positions. If thereare three substrates, for example, one substrate can differ from thesecond at a single position, and the second can differ from the third ata different single position. The starting DNA segments can be naturalvariants of each other, for example, allelic or species variants. Thesegments can also be from nonallelic genes showing some degree ofstructural and usually functional relatedness (e.g., different geneswithin a superfamily, such as the family of Yersinia V-antigens, forexample). The starting DNA segments can also be induced variants of eachother. For example, one DNA segment can be produced by error-prone PCRreplication of the other, the nucleic acid can be treated with achemical or other mutagen, or by substitution of a mutagenic cassette.Induced mutants can also be prepared by propagating one (or both) of thesegments in a mutagenic strain, or by inducing an error-prone repairsystem in the cells. In these situations, strictly speaking, the secondDNA segment is not a single segment but a large family of relatedsegments. The different segments forming the starting materials areoften the same length or substantially the same length. However, thisneed not be the case; for example; one segment can be a subsequence ofanother. The segments can be present as part of larger molecules, suchas vectors, or can be in isolated form.

The starting DNA segments are recombined by any of the sequencerecombination formats provided herein to generate a diverse library ofrecombinant DNA segments. Such a library can vary widely in size fromhaving fewer than 10 to more than 10⁵, 10⁹, 10¹² or more members. Insome embodiments, the starting segments and the recombinant librariesgenerated will include full-length coding sequences and any essentialregulatory sequences, such as a promoter and polyadenylation sequence,required for expression. In other embodiments, the recombinant DNAsegments in the library can be inserted into a common vector providingsequences necessary for expression before performingscreening/selection.

Substrates for Evolution of Optimized Recombinant Antigens

The invention provides methods of obtaining recombinant polynucleotidesthat encode antigens that exhibit improved ability to induce an immuneresponse to a pathogenic agent. The methods are applicable to a widerange of pathogenic agents, including potential biological warfareagents and other organisms and polypeptides that can cause disease andtoxicity in humans and other animals. The following examples are merelyillustrative, and not limiting.

1. Bacterial Pathogens and Toxins

In some embodiments, the methods of the invention are applied tobacterial pathogens, as well as to toxins produced by bacteria and otherorganisms. One can use the methods to obtain recombinant polypeptidesthat can induce an immune response against the pathogen, as well asrecombinant toxins that are less toxic than native toxin polypeptides.Often, the polynucleotides of interest encode polypeptides that arepresent on the surface of the pathogenic organism.

Among the pathogens for which the methods of the invention are usefulfor producing protective immunogenic recombinant polypeptides are theYersinia species. Yersinia pestis, the causative agent of plague, is oneof the most virulent bacteria known with LD₅₀ values in mouse of lessthan 10 bacteria. The pneumonic form of the disease is readily spreadbetween humans by aerosol or infectious droplets and can be lethalwithin days. A particularly preferred target for obtaining a recombinantpolypeptide that can protect against Yersinia infection is the Vantigen, which is a 37 kDa virulence factor, induces protective immuneresponses and is currently being evaluated as a subunit vaccine(Brubaker (1991) Current Investigations of the Microbiology of Yersinae,12: 127). The V-antigen alone is not toxic, but Y. pestis isolates thatlack the V-antigen are avirulent. The Yersinia V-antigen has beensuccessfully produced in E. coli by several groups (Leary et al. (1995)Infect. Immun. 3: 2854). Antibodies that recognize the V-antigen canprovide passive protection against homologous strains, but not againstheterologous strains. Similarly, immunization with purified V antigenprotects against only homologous strains. To obtain cross-protectiverecombinant V antigen, in a preferred embodiment, V antigen genes fromvarious Yersinia species are subjected to family shuffling. The genesencoding the V antigen from Y. pestis, Y. enterocolitica, and Y.pseudotuberculosis, for example, are 92-99% identical at the DNA level,making them ideal for optimization using family shuffling according tothe methods of the invention. After shuffling, the library ofrecombinant nucleic acids is screened and/or selected for those thatencode recombinant V antigen polypeptides that can induce an improvedimmune response and/or have greater cross-protectivity.

Bacillus anthracis, the causative agent of anthrax, is another exampleof a bacterial target against which the methods of the invention areuseful. The anthrax protective antigen (PA) provides protective immuneresponses in test animals, and antibodies against PA also provide someprotection. However, the immunogenicity of PA is relatively poor, somultiple injections are typically required when wild-type PA is used.Co-vaccination with lethal factor (LF) can improve the efficacy ofwild-type PA vaccines, but toxicity is a limiting factor. Accordinglythe DNA shuffling and antigen library immunization methods of theinvention can be used to obtain nontoxic LF. Polynucleotides that encodeLF from various B. anthracis strains are subjected to family shuffling.The resulting library of recombinant LF nucleic acids can then bescreened to identify those that encode recombinant LF polypeptides thatexhibit reduced toxicity. For example, one can inoculate tissue culturecells with the recombinant LF polypeptides in the presence of PA andselect those clones for which the cells survive. If desired, one canthen backcross the nontoxic LF polypeptides to retain the immunogenicepitopes of LF. Those that are selected through the first screen canthen be subjected to a secondary screen. For example, one can test forthe ability of the recombinant nontoxic LF polypeptides to induce animmune response (e.g., CTL or antibody response) in a test animal suchas mice. In preferred embodiments, the recombinant nontoxic LFpolypeptides are then tested for ability to induce protective immunityin test animals against challenge by different strains of B. anthracis.

The protective antigen (PA) of B. anthracis is also a suitable targetfor the methods of the invention. PA-encoding nucleic acids from variousstrains of B. anthracis are subjected to DNA shuffling. One can thenscreen for proper folding in, for example, E. coli, using polyclonalantibodies. Screening for ability to induce broad-spectrum antibodies ina test animal is also typically used, either alone or in addition to apreliminary screening method. In presently preferred embodiments, thoserecombinant polynucleotides that exhibit the desired properties can bebackcrossed so that the immunogenic epitopes are maintained. Finally,the selected recombinants are tested for ability to induce protectiveimmunity against different strains of B. anthracis in a test animal.

The Staphylococcus aureus and Streptococcus toxins are another exampleof a target polypeptide that can be altered using the methods of theinvention. Strains of Staphylococcus aureus and group A Streptococci areinvolved in a range of diseases, including food poisoning, toxic shocksyndrome, scarlet fever and various autoimmune disorders. They secrete avariety of toxins, which include at least five cytolytic toxins, acoagulase, and a variety of enterotoxins. The enterotoxins areclassified as superantigens in that they crosslink MHC class IImolecules with T cell receptors to cause a constitutive T cellactivation (Fields et al. (1996) Nature 384: 188). This results in theaccumulation of pathogenic levels of cytokines that can lead to multipleorgan failure and death. At least thirty related, yet distinctenterotoxins have been sequenced and can be phylogenetically groupedinto families. Crystal structures have been obtained for several membersalone and in complex with MHC class II molecules. Certain mutations inthe MHC class II-binding site of the toxins strongly reduce theirtoxicity and can form the basis of attenuated vaccines (Woody et al.(1997) Vaccine 15: 133). However, a successful immune response to onetype of toxin may provide protection against closely related familymembers, whereas little protection against toxins from the otherfamilies is observed. Family shuffling of enterotoxin genes from variousfamily members can be used to obtain recombinant toxin molecules thathave reduced toxicity and can induce a cross-protective immune response.Shuffled antigens can also be screened to identify antigens that elicitneutralizing antibodies in an appropriate animal model such as mouse ormonkey. Examples of such assays can include ELISA formats in which theelicited antibodies prevent binding of the enterotoxin to the MHCcomplex and/or T cell receptors on cells or purified forms. These assayscan also include formats where the added antibodies would prevent Tcells from being cross-linked to appropriate antigen presenting cells.

Cholera is an ancient, potentially lethal disease caused by thebacterium Vibrio cholerae and an effective vaccine for its prevention isstill unavailable. Much of the pathogenesis of this disease is caused bythe cholera enterotoxin. Ingestion of microgram quantities of choleratoxin can induce severe diarrhea causing loss of tens of liters offluid. Cholera toxin is a complex of a single catalytic A subunit with apentameric ring of identical B subunits. Each subunit is inactive on itsown. The B subunits bind to specific ganglioside receptors on thesurface of intestinal epithelial cells and trigger the entry of the Asubunit into the cell. The A subunit ADP-ribosylates a regulatory Gprotein initiating a cascade of events causing a massive, sustained flowof electrolytes and water into the intestinal lumen resulting in extremediarrhea.

The B subunit of cholera toxin is an attractive vaccine target for anumber of reasons. It is a major target of protective antibodiesgenerated during cholera infection and contains the epitopes forantitoxin neutralizing antibodies. It is nontoxic without the A subunit,is orally effective, and stimulates production of a strong IgA-dominatedgut mucosal immune response, which is essential in protection againstcholera and cholera toxin. The B subunit is also being investigated foruse as an adjuvant in other vaccine preparations, and therefore, evolvedtoxins may provide general improvements for a variety of differentvaccines. The heat-labile enterotoxins (LT) from enterotoxigenic E. colistrains are structurally related to cholera toxin and are 75% identicalat the DNA sequence level. To obtain optimized recombinant toxinmolecules that exhibit reduced toxicity and increased ability to inducean immune response that is protective against V. cholerae and E. coli,the genes that encode the related toxins are subjected to DNA shuffling.

The recombinant toxins are then tested for one or more of a severaldesirable traits. For example, one can screen for improvedcross-reactivity of antibodies raised against the recombinant toxinpolypeptides, for lack of toxicity in a cell culture assay, and forability to induce a protective immune response against the pathogensand/or against the toxins themselves. The shuffled clones can beselected by phage display and/or screened by phage ELISA and ELISAassays for the presence of epitopes from the different serotypes.Variant proteins with multiple epitopes can then be purified and used toimmunize mice or other test animal. The animal serum is then assayed forantibodies to the different B chain subtypes and variants that elicit abroad cross-reactive response will be evaluated further in a virulentchallenge model. The E. coli and V. cholerae toxins can also act asadjuvants that are capable of enhancing mucosal immunity and oraldelivery of vaccines and proteins. Accordingly, one can test the libraryof recombinant toxins for enhancement of the adjuvant activity.

Shuffled antigens can also be screened for improved expression levelsand stability of the B chain pentamer, which may be less stable thanwhen in the presence of the A chain in the hexameric complex. Additionof a heat treatment step or denaturing agents such as salts, urea,and/or guanidine hydrochloride can be included prior to ELISA assays tomeasure yields of correctly folded molecules by appropriate antibodies.It is sometimes desirable to screen for stable monomeric B chainmolecules, in an ELISA format, for example, using antibodies that bindmonomeric, but not pentameric B chains. Additionally, the ability ofshuffled antigens to elicit neutralizing antibodies in an appropriateanimal model such as mouse or monkey can be screened. For example,antibodies that bind to the B chain and prevent its binding to itsspecific ganglioside receptors on the surface of intestinal epithelialcells may prevent disease. Similarly antibodies that bind to the B chainand prevent its pentamerization or block A chain binding may be usefulin preventing disease.

The bacterial antigens that can be improved by DNA shuffling for use asvaccines also include, but are not limited to, Helicobacter pyloriantigens CagA and VacA (Blaser (1996) Aliment. Pharmacol. Ther. 1: 73-7;Blaser and Crabtree (1996) Am. J. Clin. Pathol. 106: 565-7; Censini etal. (1996) Proc. Nat'l. Acad. Sci. USA 93: 14648-14643). Other suitableH. pylori antigens include, for example, four immunoreactive proteins of45-65 kDa as reported by Chatha et al. (1997) Indian J. Med. Res. 105:170-175 and the H. pylori GroES homologue (HspA) (Kansau et al. (1996)Mol. Microbiol. 22: 1013-1023. Other suitable bacterial antigensinclude, but are not limited to, the 43-kDa and the fimbrilin (41 kDa)proteins of P. gingivalis (Boutsl et al. (1996) Oral Microbiol. Immunol.11: 236-241); pneumococcal surface protein A (Briles et al. (1996) Ann.NY Acad. Sci. 797: 118-126); Chlamydia psittaci antigens, 80-90 kDaprotein and 110 kDa protein (Buendia et al. (1997) FEMS Microbiol. Lett.150: 113-9); the chlamydial exoglycolipid antigen (GLXA) (Whittum-Hudsonet al. (1996) Nature Med. 2: 1116-1121); Chlamydia pneumoniaespecies-specific antigens in the molecular weight ranges 92-98, 51-55,43-46 and 31.5-33 kDa and genus-specific antigens in the ranges 12, 26and 65-70 kDa (Halme et al. (1997) Scand. J. Immunol. 45: 378-84);Neisseria gonorrhoeae (GC) or Escherichia coli phase-variable opacity(Opa) proteins (Chen and Gotschlich (1996) Proc. Nat'l. Acad. Sci. USA93: 14851-14856), any of the twelve immunodominant proteins ofSchistosoma mansoni (ranging in molecular weight from 14 to 208 kDa) asdescribed by Cutts and Wilson (1997) Parasitology 114: 245-55; the17-kDa protein antigen of Brucella abortus (De Mot et al. (1996) Curr.Microbiol. 33: 26-30); a gene homolog of the 17-kDa protein antigen ofthe Gram-negative pathogen Brucella abortus identified in thenocardioform actinomycete Rhodococcus sp. NI86/21 (De Mot et al. (1996)Curr. Microbiol. 33: 26-30); the staphylococcal enterotoxins (SEs) (Woodet al. (1997) FEMS Immunol. Med. Microbiol. 17: 1-10), a 42-kDa M.hyopneumoniae NrdF ribonucleotide reductase R2 protein or 15-kDa subunitprotein of M. hyopneumoniae (Fagan et al. (1997) Infect. Immun. 65:2502-2507), the meningococcal antigen PorA protein (Feavers et al.(1997) Clin. Diagn. Lab. Immunol. 3: 444-50); pneumococcal surfaceprotein A (PspA) (McDaniel et al. (1997) Gene Ther. 4: 375-377); F.tularensis outer membrane protein FopA (Fulop et al. (1996) FEMSImmunol. Med. Microbiol. 13: 245-247); the major outer membrane proteinwithin strains of the genus Actinobacillus (Hartmann et al. (1996)Zentralbl. Bakteriol. 284: 255-262); p60 or listeriolysin (Hly) antigenof Listeria monocytogenes (Hess et al. (1996) Proc. Nat'l. Acad. Sci.USA 93: 1458-1463); flagellar (G) antigens observed on Salmonellaenteritidis and S. pullorum (Holt and Chaubal (1997) J. Clin. Microbiol.35: 1016-1020); Bacillus anthracis protective antigen (PA) (Ivins et al.(1995) Vaccine 13: 1779-1784); Echinococcus granulosus antigen 5 (Joneset al. (1996) Parasitology 113: 213-222); the rol genes of Shigelladysenteriae 1 and Escherichia coli K-12 (Klee et al. (1997) J.Bacteriol. 179: 2421-2425); cell surface proteins Rib and alpha of groupB streptococcus (Larsson et al. (1996) Infect. Immun. 64: 3518-3523);the 37 kDa secreted polypeptide encoded on the 70 kb virulence plasmidof pathogenic Yersinia spp. (Leary et al. (1995) Contrib. Microbiol.Immunol. 13: 216-217 and Roggenkamp et al. (1997) Infect. Immun. 65:446-51); the OspA (outer surface protein A) of the Lyme diseasespirochete Borrelia burgdorferi (Li et al. (1997) Proc. Nat'l. Acad.Sci. USA 94: 3584-3589, Padilla et al. (1996) J. Infect. Dis. 174:739-746, and Wallich et al. (1996) Infection 24: 396-397); the Brucellamelitensis group 3 antigen gene encoding Omp28 (Lindler et al. (1996)Infect. Immun. 64: 2490-2499); the PAc antigen of Streptococcus mutans(Murakami et al. (1997) Infect. Immun. 65: 794-797); pneumolysin,Pneumococcal neuraminidases, autolysin, hyaluronidase, and the 37 kDapneumococcal surface adhesin A (Paton et al. (1997) Microb. Drug Resist.3: 1-10); 29-32, 41-45, 63-71×10(3) MW antigens of Salmonella typhi(Perez et al. (1996) Immunology 89: 262-267); K-antigen as a marker ofKlebsiella pneumoniae (Priamukhina and Morozova (1996) Klin. Lab. Diagn.47-9); nocardial antigens of molecular mass approximately 60, 40, 20 and15-10 kDa (Prokesova et al. (1996) Int. J. Immunopharmacol. 18:661-668); Staphylococcus aureus antigen ORF-2 (Rieneck et al. (1997)Biochim Biophys Acta 1350: 128-132); GlpQ antigen of Borrelia hermsii(Schwan et al. (1996) J. Clin. Microbiol. 34: 2483-2492); choleraprotective antigen (CPA) (Sciortino (1996) J. Diarrhoeal Dis. Res. 14:16-26); a 190-kDa protein antigen of Streptococcus mutans (Senpuku etal. (1996) Oral Microbiol. Immunol. 11: 121-128); Anthrax toxinprotective antigen (PA) (Sharma et al. (1996) Protein Expr. Purif 7:33-38); Clostridium perfringens antigens and toxoid (Strom et al. (1995)Br. J. Rheumatol. 34: 1095-1096); the SEF14 fimbrial antigen ofSalmonella enteritidis (Thoms et al. (1996) Microb. Pathog. 20:235-246); the Yersinia pestis capsular antigen (F1 antigen) (Titball etal. (1997) Infect. Immun. 65:1926-1930); a 35-kilodalton protein ofMycobacterium leprae (Triccas et al. (1996) Infect. Immun. 64:5171-5177); the major outer membrane protein, CD, extracted fromMoraxella (Branhamella) catarrhalis (Yang et al. (1997) FEMS Immunol.Med. Microbiol. 17: 187-199); pH6 antigen (PsaA protein) of Yersiniapestis (Zav'yalov et al. (1996) FEMS Immunol. Med. Microbiol. 14:53-57); a major surface glycoprotein, gp63, of Leishmania major (Xu andLiew (1994) Vaccine 12: 1534-1536; Xu and Liew (1995) Immunology 84:173-176); mycobacterial heat shock protein 65, mycobacterial antigen(Mycobacterium leprae hsp65) (Lowrie et al. (1994) Vaccine 12:1537-1540; Ragno et al. (1997) Arthritis Rheum. 40: 277-283; Silva(1995) Braz. J. Med. Biol. Res. 28: 843-851); Mycobacterium tuberculosisantigen 85 (Ag85) (Huygen et al. (1996) Nat. Med. 2: 893-898); the 45/47kDa antigen complex (APA) of Mycobacterium tuberculosis, M. bovis andBCG (Horn et al. (1996) J. Immunol. Methods 197: 151-159); themycobacterial antigen, 65-kDa heat shock protein, hsp65 (Tascon et al.(1996) Nat. Med. 2: 888-892); the mycobacterial antigens MPB64, MPB70,MPB57 and alpha antigen (Yamada et al. (1995) Kekkaku 70: 639-644); theM tuberculosis 38 kDa protein (Vorderneier et al. (1995) Vaccine 13:1576-1582); the MPT63, MPT64 and MPT-59 antigens from Mycobacteriumtuberculosis (Manca et al. (1997) Infect. Immun. 65: 16-23; Oettinger etal. (1997) Scand. J. Immunol. 45: 499-503; Wilcke et al. (1996) Tuber.Lung Dis. 77: 250-256); the 35-kilodalton protein of Mycobacteriumleprae (Triccas et al. (1996) Infect. Immun. 64: 5171-5177); the ESAT-6antigen of virulent mycobacteria (Brandt et al. (1996) J. Immunol. 157:3527-3533; Pollock and Andersen (1997) J. Infect. Dis. 175: 1251-1254);Mycobacterium tuberculosis 16-kDa antigen (Hsp16.3) (Chang et al. (1996)J. Biol. Chem. 271: 7218-7223); and the 18-kilodalton protein ofMycobacterium leprae (Baumgart et al. (1996) Infect. Immun. 64:2274-2281).

2. Viral Pathogens

The methods of the invention are also useful for obtaining recombinantnucleic acids and polypeptides that have enhanced ability to induce animmune response against viral pathogens. While the bacterialrecombinants described above are typically administered in polypeptideform, recombinants that confer viral protection are preferablyadministered in nucleic acid form, as genetic vaccines.

One illustrative example is the Hantaan virus. Glycoproteins of thisvirus typically accumulate at the membranes of the Golgi apparatus ofinfected cells. This poor expression of the glycoprotein prevents thedevelopment of efficient genetic vaccines against these viruses. Themethods of the invention solve this problem by performing DNA shufflingon nucleic acids that encode the glycoproteins and identifying thoserecombinants that exhibit enhanced expression in a host cell, and/or forimproved immunogenicity when administered as a genetic vaccine. Aconvenient screening method for these methods is to express therecombinant polynucleotides as fusion proteins to PIG, which results indisplay of the polypeptides on the surface of the host cell (Whitehornet al. (1995) Biotechnology (N Y) 13:1215-9). Fluorescence-activatedcell sorting is then used to sort and recover those cells that expressan increased amount of the antigenic polypeptide on the cell surface.This preliminary screen can be followed by immunogenicity tests inmammals, such as mice. Finally, in preferred embodiments, thoserecombinant nucleic acids are tested as genetic vaccines for theirability to protect a test animal against challenge by the virus.

The flaviviruses are another example of a viral pathogen for which themethods of the invention are useful for obtaining a recombinantpolypeptide or genetic vaccine that is effective against a viralpathogen. The flaviviruses consist of three clusters of antigenicallyrelated viruses: Dengue 1-4 (62-77% identity), Japanese, St. Louis andMurray Valley encephalitis viruses (75-82% identity), and the tick-borneencephalitis viruses (77-96% identity). Dengue virus can induceprotective antibodies against SLE and Yellow fever (40-50% identity),but few efficient vaccines are available. To obtain genetic vaccines andrecombinant polypeptides that exhibit enhanced cross-reactivity andimmunogenicity, the polynucleotides that encode envelope proteins ofrelated viruses are subjected to DNA shuffling. The resultingrecombinant polynucleotides can be tested, either as genetic vaccines orby using the expressed polypeptides, for ability to induce a broadlyreacting neutralizing antibody response. Finally, those clones that arefavorable in the preliminary screens can be tested for ability toprotect a test animal against viral challenge.

Viral antigens that can be evolved by DNA shuffling for improvedactivity as vaccines include, but are not limited to, influenza A virusN2 neuraminidase (Kilbourne et al. (1995) Vaccine 13: 1799-1803); Denguevirus envelope (E) and premembrane (prM) antigens (Feighny et al. (1994)Am. J. Trop. Med. Hyg. 50: 322-328; Putnak et al. (1996) Am. J. Trop.Med. Hyg. 55: 504-10); HIV antigens Gag, Pol, Vif and Nef (Vogt et al.(1995) Vaccine 13: 202-208); HIV antigens gp120 and gp160 (Achour et al.(1995) Cell. Mol. Biol. 41: 395-400; Hone et al. (1994) Dev. Biol.Stand. 82: 159-162); gp41 epitope of human immunodeficiency virus(Eckhart et al. (1996) J. Gen. Virol. 77: 2001-2008); rotavirus antigenVP4 (Mattion et al. (1995) J. Virol. 69: 5132-5137); the rotavirusprotein VP7 or VP7sc (Emslie et al. (1995) J. Virol. 69: 1747-1754; Xuet al. (1995) J. Gen. Virol. 76: 1971-1980); herpes simplex virus (HSV)glycoproteins gB, gC, gD, gE, gG, gH, and gI (Fleck et al. (1994) Med.Microbiol. Immunol. (Berl) 183: 87-94 [Mattion, 1995]; Ghiasi et al.(1995) Invest. Ophthalmol. Vis. Sci. 36: 1352-1360; McLean et al. (1994)J. Infect. Dis. 170: 1100-1109); immediate-early protein ICP47 of herpessimplex virus-type 1 (HSV-1) (Banks et al. (1994) Virology 200:236-245); immediate-early (IE) proteins ICP27, ICPO, and ICP4 of herpessimplex virus (Manickan et al. (1995) J. Virol. 69: 4711-4716);influenza virus nucleoprotein and hemagglutinin (Deck et al. (1997)Vaccine 15: 71-78; Fu et al. (1997) J. Virol. 71: 2715-2721); B19parvovirus capsid proteins VP1 (Kawase et al. (1995) Virology 211:359-366) or VP2 (Brown et al. (1994) Virology 198: 477-488); Hepatitis Bvirus core and e antigen (Schodel et al. (1996) Intervirology 39:104-106); hepatitis B surface antigen (Shiau and Murray (1997) J. Med.Virol. 51: 159-166); hepatitis B surface antigen fused to the coreantigen of the virus (Id.); Hepatitis B virus core-preS2 particles(Nemeckova et al. (1996) Acta Virol. 40: 273-279); HBV preS2-S protein(Kutinova et al. (1996) Vaccine 14: 1045-1052); VZV glycoprotein I(Kutinova et al. (1996) Vaccine 14: 1045-1052); rabies virusglycoproteins (Xiang et al. (1994) Virology 199: 132-140; Xuan et al.(1995) Virus Res. 36: 151-161) or ribonucleocapsid (Hooper et al. (1994)Proc. Nat'l. Acad. Sci. USA 91: 10908-10912); human cytomegalovirus(HCMV) glycoprotein B (UL55) (Britt et al. (1995) J. Infect. Dis. 171:18-25); the hepatitis C virus (HCV) nucleocapsid protein in a secretedor a nonsecreted form, or as a fusion protein with the middle (pre-S2and S) or major (S) surface antigens of hepatitis B virus (HBV)(Inchauspe et al. (1997) DNA Cell Biol. 16: 185-195; Major et al. (1995)J. Virol. 69: 5798-5805); the hepatitis C virus antigens: the coreprotein (pC); E1 (pE1) and E2 (pE2) alone or as fusion proteins (Saitoet al. (1997) Gastroenterology 112: 1321-1330); the gene encodingrespiratory syncytial virus fusion protein (PFP-2) (Falsey and Walsh(1996) Vaccine 14: 1214-1218; Piedra et al. (1996) Pediatr. Infect. Dis.J. 15: 23-31); the VP6 and VP7 genes of rotaviruses (Choi et al. (1997)Virology 232: 129-138; Jin et al. (1996) Arch. Virol. 141: 2057-2076);the E1, E2, E3, E4, E5, E6 and E7 proteins of human papillomavirus(Brown et al. (1994) Virology 201: 46-54; Dillner et al. (1995) CancerDetect. Prev. 19: 381-393; Krul et al. (1996) Cancer Immunol.Immunother. 43: 44-48; Nakagawa et al. (1997) J. Infect. Dis. 175:927-931); a human T-lymphotropic virus type I gag protein (Porter et al.(1995) J. Med. Virol. 45: 469-474); Epstein-Barr virus (EBV) gp340(Mackett et al. (1996) J. Med. Virol. 50: 263-271); the Epstein-Barrvirus (EBV) latent membrane protein LMP2 (Lee et al. (1996) Eur. J.Immunol. 26: 1875-1883); Epstein-Barr virus nuclear antigens 1 and 2(Chen and Cooper (1996) J. Virol. 70: 4849-4853; Khanna et al. (1995)Virology 214: 633-637); the measles virus nucleoprotein (N) (Fooks etal. (1995) Virology 210: 456-465); and cytomegalovirus glycoprotein gB(Marshall et al. (1994) J. Med. Virol. 43: 77-83) or glycoprotein gH(Rasmussen et al. (1994) J. Infect. Dis. 170: 673-677).

3. Parasites

Antigens from parasites can also be optimized by the methods of theinvention. These include, but are not limited to, the schistosomegut-associated antigens CAA (circulating anodic antigen) and CCA(circulating cathodic antigen) in Schistosoma mansoni, S. haematobium orS. japonicum (Deelder et al. (1996) Parasitology 112: 21-35); a multipleantigen peptide (MAP) composed of two distinct protective antigensderived from the parasite Schistosoma mansoni (Ferru et al. (1997)Parasite Immunol. 19: 1-11); Leishmania parasite surface molecules(Lezama-Davila (1997) Arch. Med. Res. 28: 47-53); third-stage larval(L3) antigens of L. loa (Akue et al. (1997) J. Infect. Dis. 175:158-63); the genes, Tams1-1 and Tams1-2, encoding the 30- and 32-kDamajor merozoite surface antigens of Theileria annulata (Ta) (d'Oliveiraet al. (1996) Gene 172: 33-39); Plasmodium falciparum merozoite surfaceantigen 1 or 2 (al-Yaman et al. (1995) Trans. R. Soc. Trop. Med. Hyg.89: 555-559; Beck et al. (1997) J. Infect. Dis. 175: 921-926; Rzepczyket al. (1997) Infect. Immun. 65: 1098-1100); circumsporozoite (CS)protein-based B-epitopes from Plasmodium berghei, (PPPPNPND)₂ andPlasmodium yoelii, (QGPGAP)₃QG, along with a P. berghei T-helper epitopeKQIRDSITEEWS (Reed et al. (1997) Vaccine 15: 482-488); NYVAC-Pf7 encodedPlasmodium falciparum antigens derived from the sporozoite(circumsporozoite protein and sporozoite surface protein 2), liver(liver stage antigen 1), blood (merozoite surface protein 1, serinerepeat antigen, and apical membrane antigen 1), and sexual (25-kDasexual-stage antigen) stages of the parasite life cycle were insertedinto a single NYVAC genome to generate NYVAC-Pf7 (Tine et al. (1996)Infect. Immun. 64: 3833-3844); Plasmodium falciparum antigen Pfs230(Williamson et al. (1996) Mol. Biochem. Parasitol. 78: 161-169);Plasmodium falciparum apical membrane antigen (AMA-1) (Lal et al. (1996)Infect. Immun. 64: 1054-1059); Plasmodium falciparum proteins Pfs28 andPfs25 (Duffy and Kaslow (1997) Infect. Immun. 65: 1109-1113); Plasmodiumfalciparum merozoite surface protein, MSP1 (Hui et al. (1996) Infect.Immun. 64: 1502-1509); the malaria antigen Pf332 (Ahlborg et al. (1996)Immunology 88: 630-635); Plasmodium falciparum erythrocyte membraneprotein 1 (Baruch et al. (1995) Proc. Nat'l. Acad. Sci. USA 93:3497-3502; Baruch et al. (1995) Cell 82: 77-87); Plasmodium falciparummerozoite surface antigen, PfMSP-1 (Egan et al. (1996) J. Infect. Dis.173: 765-769); Plasmodium falciparum antigens SERA, EBA-175, RAP1 andRAP2 (Riley (1997) J. Pharm. Pharmacol. 49: 21-27); Schistosomajaponicum paramyosin (Sj97) or fragments thereof (Yang et al. (1995)Biochem. Biophys. Res. Commun. 212: 1029-1039); and Hsp70 in parasites(Maresca and Kobayashi (1994) Experientia 50: 1067-1074).

4. Allergy

The invention also provides methods of obtaining reagents that areuseful for treating allergy. In one embodiment, the methods involvemaking a library of recombinant polynucleotides that encode an allergen,and screening the library to identify those recombinant polynucleotidesthat exhibit improved properties when used as immunotherapeutic reagentsfor treating allergy. For example, specific immunotherapy of allergyusing natural antigens carries a risk of inducing anaphylaxis, which canbe initiated by cross-linking of high-affinity IgE receptors on mastcells. Therefore, allergens that are not recognized by pre-existing IgEare desirable. The methods of the invention provide methods by which onecan obtain such allergen variants. Another improved property of interestis induction of broader immune responses, increased safety and efficacy.

Synthesis of polyclonal and allergen-specific IgE requires multipleinteractions between B cells, T cells and professionalantigen-presenting cells (APC). Activation of naive, unprimed B cells isinitiated when specific B cells recognize the allergen by cell surfaceimmunoglobulin (sIg). However, costimulatory molecules expressed byactivated T cells in both soluble and membrane-bound forms are necessaryfor differentiation of B cells into IgE-secreting plasma cells.Activation of T helper cells requires recognition of an antigenicpeptide in the context of MHC class II molecules on the plasma membraneof APC, such as monocytes, dendritic cells, Langerhans cells or primed Bcells. Professional APC can efficiently capture the antigen and thepeptide-MHC class II complexes are formed in a post-Golgi, proteolyticintracellular compartment and subsequently exported to the plasmamembrane, where they are recognized by T cell receptor (TCR) (Whitton(1998) Curr. Top. Microbiol. Immunol. 232: 1-13). In addition, activatedB cells express CD80 (B7-1) and CD86 (B7-2, B70), which are the counterreceptors for CD28 and which provide a costimulatory signal for T cellactivation resulting in T cell proliferation and cytokine synthesis.Since allergen-specific T cells from atopic individuals generally belongto the T_(H)2 cell subset, activation of these cells also leads toproduction of IL-4 and IL-13, which, together with membrane-boundcostimulatory molecules expressed by activated T helper cells, direct Bcell differentiation into IgE-secreting plasma cells.

Mast cells and eosinophils are key cells in inducing allergic symptomsin target organs. Recognition of specific antigen by IgE bound tohigh-affinity IgE receptors on mast cells, basophils or eosinophilsresults in crosslinking of the receptors leading to degranulation of thecells and rapid release of mediator molecules, such as histamine,prostaglandins and leukotrienes, causing allergic symptoms.

Immunotherapy of allergic diseases currently includeshyposensibilization treatments using increasing doses of allergeninjected to the patient. These treatments result skewing of immuneresponses towards T_(H)1 phenotype and increase the ratio of IgG/IgEantibodies specific for allergens. Because these patients havecirculating IgE antibodies specific for the allergens, these treatmentsinclude significant risk of anaphylactic reactions. In these reactions,free circulating allergen is recognized by IgE molecules bound tohigh-affinity IgE receptors on mast cells and eosinophils. Recognitionof the allergen results in crosslinking of the receptors leading torelease of mediators, such as histamine, prostaglandins, andleukotrienes, which cause the allergic symptoms, and occasionallyanaphylactic reactions. Other problems associated withhyposensibilization include low efficacy and difficulties in producingallergen extracts reproducibly.

The methods of the invention provide a means to obtain allergens that,when used in genetic vaccines, provide a means of circumventing theproblems that have limited the usefulness of previously knownhyposensibilization treatments. For example, by expressing antigens onthe surface of cells, such as muscle cells, the risk of anaphylacticreactions is significantly reduced. This can be conveniently achieved byusing genetic vaccine vectors that encode transmembrane forms ofallergens. The allergens can also be modified in such a way that theyare efficiently expressed in transmembrane forms, further reducing therisk of anaphylactic reactions. Another advantage provided by the use ofgenetic vaccines for hyposensibilization is that the genetic vaccinescan include cytokines and accessory molecules which further direct theimmune responses towards the T_(H)1 phenotype, thus reducing the amountof IgE antibodies produced and increasing the efficacy of thetreatments. To further reduce IgE production, one can administer theshuffled allergens using vectors that have been evolved to induceprimarily IgG and IgM responses, with little or no IgE response (see,e.g., U.S. patent application Ser. No. 09/021,769, filed Feb. 11, 1998).

In these methods, polynucleotides encoding known allergens, or homologsor fragments thereof (e.g., immunogenic peptides) are inserted into DNAvaccine vectors and used to immunize allergic and asthmatic individuals.Alternatively, the shuffled allergens are expressed in manufacturingcells, such as E. coli or yeast cells, and subsequently purified andused to treat the patients or prevent allergic disease. DNA shuffling orother recombination method can be used to obtain allergens that activateT cells but cannot induce anaphylactic reactions. For example, a libraryof recombinant polynucleotides that encode allergen variants can beexpressed in cells, such as antigen presenting cells, which are thancontacted with PBMC or T cell clones from atopic patients. Those librarymembers that efficiently activate T_(H) cells from the atopic patientscan be identified by assaying for T cell proliferation, or by cytokinesynthesis (e.g., synthesis of IL-2, IL-4, IFN-γ. Those recombinantallergen variants that are positive in the in vitro tests can then besubjected to in vivo testing.

Examples of allergies that can be treated include, but are not limitedto, allergies against house dust mite, grass pollen, birch pollen,ragweed pollen, hazel pollen, cockroach, rice, olive tree pollen, fungi,mustard, bee venom. Antigens of interest include those of animals,including the mite (e.g., Dermatophagoides pteronyssinnus,Dermatophagoides farinae, Blomia tropicalis), such as the allergens derp1 (Scobie et al. (1994) Biochem. Soc. Trans. 22: 448S; Yssel et al.(1992) J. Immunol. 148: 738-745), der p2 (Chua et al. (1996) Clin. Exp.Allergy 26: 829-837), der p3 (Smith and Thomas (1996) Clin. Exp. Allergy26: 571-579), der p5, der p V (Lin et al. (1994) J. Allergy Clin.Immunol. 94: 989-996), der p6 (Bennett and Thomas (1996) Clin. Exp.Allergy 26: 1150-1154), der p 7 (Shen et al. (1995) Clin. Exp. Allergy25: 416-422), der f2 (Yuuki et al. (1997) Int. Arch. Allergy Immunol.112: 44-48), der f3 (Nishiyama et al. (1995) FEBS Lett. 377: 62-66), derf7 (Shen et al. (1995) Clin. Exp. Allergy 25: 1000-1006); Mag 3(Fujikawa et al. (1996) Mol. Immunol. 33: 311-319). Also of interest asantigens are the house dust mite allergens Tyr p2 (Eriksson et al.(1998) Eur. J. Biochem. 251: 443-447), Lep d1 (Schmidt et al. (1995)FEBS Lett. 370: 11-14), and glutathione S-transferase (O'Neill et al.(1995) Immunol Lett. 48: 103-107); the 25,589 Da, 219 amino acidpolypeptide with homology with glutathione S-transferases (O'Neill etal. (1994) Biochim. Biophys. Acta. 1219: 521-528); Blo t 5 (Arruda etal. (1995) Int. Arch. Allergy Immunol. 107: 456-457); bee venomphospholipase A2 (Carballido et al. (1994) J. Allergy Clin. Immunol. 93:758-767; Jutel et al. (1995) J. Immunol. 154: 4187-4194); bovinedermal/dander antigens BDA 11 (Rautiainen et al. (1995) J. Invest.Dermatol. 105: 660-663) and BDA20 (Mantyjarvi et al. (1996) J. AllergyClin. Immunol. 97: 1297-1303); the major horse allergen Equ c1 (Gregoireet al. (1996) J. Biol. Chem. 271: 32951-32959); Jumper ant M. pilosulaallergen Myr p I and its homologous allergenic polypeptides Myr p2(Donovan et al. (1996) Biochem. Mol. Biol. Int. 39: 877-885); 1-13, 14,16 kD allergens of the mite Blomia tropicalis (Caraballo et al. (1996)J. Allergy Clin. Immunol. 98: 573-579); the cockroach allergens Bla gBd90K (Helm et al. (1996) J. Allergy Clin. Immunol. 98: 172-80) and Blag 2 (Arruda et al. (1995) J. Biol. Chem. 270: 19563-19568); thecockroach Cr-PI allergens (Wu et al. (1996) J. Biol. Chem. 271:17937-17943); fire ant venom allergen, Sol i 2 (Schmidt et al. (1996) J.Allergy Clin. Immunol. 98: 82-88); the insect Chironomus thummi majorallergen Chi t 1-9 (Kipp et al. (1996) Int. Arch. Allergy Immunol. 110:348-353); dog allergen Can f 1 or cat allergen Fel d 1 (Ingram et al.(1995) J. Allergy Clin. Immunol. 96: 449-456); albumin, derived forexample, from horse, dog or cat (Goubran Botros et al. (1996) Immunology88: 340-347); deer allergens with the molecular mass of 22 kD, 25 kD or60 kD (Spitzauer et al. (1997) Clin. Exp. Allergy 27: 196-200); and the20 kd major allergen of cow (Ylonen et al. (1994) J. Allergy Clin.Immunol. 93: 851-858).

Pollen and grass allergens are also useful in vaccines, particularlyafter optimization of the antigen by the methods of the invention. Suchallergens include, for example, Hor v9 (Astwood and Hill (1996) Gene182: 53-62, Lig v1 (Batanero et al. (1996) Clin. Exp. Allergy 26:1401-1410); Lol p 1 (Muller et al. (1996) Int. Arch. Allergy Immunol.109: 352-355), Lol p II (Tamborini et al. (1995) Mol. Immunol. 32:505-513), Lol pVA, Lol pVB (Ong et al. (1995) Mol. Immunol. 32:295-302), Lol p 9 (Blaher et al. (1996) J. Allergy Clin. Immunol. 98:124-132); Par J I (Costa et al. (1994) FEBS Lett. 341: 182-186; Sallustoet al. (1996) J. Allergy Clin. Immunol. 97: 627-637), Par j 2.0101 (Duroet al. (1996) FEBS Lett. 399: 295-298); Bet v1 (Faber et al. (1996) J.Biol. Chem. 271: 19243-19250), Bet v2 (Rihs et al. (1994) Int. Arch.Allergy Immunol. 105: 190-194); Dac g3 (Guerin-Marchand et al. (1996)Mol. Immunol. 33: 797-806); Phl p 1 (Petersen et al. (1995) J. AllergyClin. Immunol. 95: 987-994), Phl p 5 (Muller et al. (1996) Int. Arch.Allergy Immunol. 109: 352-355), Phl p 6 (Petersen et al. (1995) Int.Arch. Allergy Immunol. 108: 55-59); Cry j I (Sone et al. (1994) Biochem.Biophys. Res. Commun. 199: 619-625), Cry j II (Namba et al. (1994) FEBSLett. 353: 124-128); Cora I (Schenk et al. (1994) Eur. J. Biochem. 224:717-722); cyn d1 (Smith et al. (1996) J. Allergy Clin. Immunol. 98:331-343), cyn d7 (Suphioglu et al. (1997) FEBS Lett. 402: 167-172); Phaa 1 and isoforms of Pha a 5 (Suphioglu and Singh (1995) Clin. Exp.Allergy 25: 853-865); Cha o 1 (Suzuki et al. (1996) Mol. Immunol. 33:451-460); profilin derived, e.g, from timothy grass or birch pollen(Valenta et al. (1994) Biochem. Biophys. Res. Commun. 199: 106-118);P0149 (Wu et al. (1996) Plant Mol. Biol. 32: 1037-1042); Ory s1 (Xu etal. (1995) Gene 164: 255-259); and Amb a V and Amb t 5 (Kim et al.(1996) Mol. Immunol. 33: 873-880; Zhu et al. (1995) J. Immunol. 155:5064-5073).

Vaccines against food allergens can also be developed using the methodsof the invention. Suitable antigens for shuffling include, for example,profilin (Rihs et al. (1994) Int. Arch. Allergy Immunol. 105: 190-194);rice allergenic cDNAs belonging to the alpha-amylase/trypsin inhibitorgene family (Alvarez et al. (1995) Biochim Biophys Acta 1251: 201-204);the main olive allergen, Ole e I (Lombardero et al. (1994) Clin ExpAllergy 24: 765-770); Sin a 1, the major allergen from mustard (GonzalezDe La Pena et al. (1996) Eur J. Biochem. 237: 827-832); parvalbumin, themajor allergen of salmon (Lindstrom et al. (1996) Scand. J. Immunol. 44:335-344); apple allergens, such as the major allergen Mal d 1(Vanek-Krebitz et al. (1995) Biochem. Biophys. Res. Commun. 214:538-551); and peanut allergens, such as Ara h I (Burks et al. (1995) J.Clin. Invest. 96:1715-1721).

The methods of the invention can also be used to develop recombinantantigens that are effective against allergies to fungi. Fungal allergensuseful in these vaccines include, but are not limited to, the allergen,Cla h III, of Cladosporium herbarum (Zhang et al. (1995) J. Immunol.154: 710-717); the allergen Psi c 2, a fungal cyclophilin, from thebasidiomycete Psilocybe cubensis (Horner et al. (1995) Int. Arch.Allergy Immunol. 107: 298-300); hsp 70 cloned from a cDNA library ofCladosporium herbarum (Zhang et al. (1996) Clin Exp Allergy 26: 88-95);the 68 kD allergen of Penicillium notatum (Shen et al. (1995) Clin. Exp.Allergy 26: 350-356); aldehyde dehydrogenase (ALDH) (Achatz et al.(1995) Mol Immunol. 32: 213-227); enolase (Achatz et al. (1995) Mol.Immunol. 32: 213-227); YCP4 (Id.); acidic ribosomal protein P2 (Id.).

Other allergens that can be used in the methods of the invention includelatex allergens, such as a major allergen (Hev b 5) from natural rubberlatex (Akasawa et al. (1996) J. Biol. Chem. 271: 25389-25393; Slater etal. (1996) J. Biol. Chem. 271: 25394-25399).

The invention also provides a solution to another shortcoming ofvaccination as a treatment for allergy and asthma. While geneticvaccination primarily induces CD8⁺ T cell responses, induction ofallergen-specific IgE responses is dependent on CD4⁺ T cells and theirhelp to B cells. T_(H)2-type cells are particularly efficient ininducing IgE synthesis because they secrete high levels of IL-4, IL-5and IL-13, which direct Ig isotype switching to IgE synthesis. IL-5 alsoinduces eosinophilia. The methods of the invention can be used todevelop recombinant antigens that efficiently induce CD4⁺ T cellresponses, and direct differentiation of these cells towards the T_(H)1phenotype.

5. Inflammatory and Autoimmune Diseases

Autoimmune diseases are characterized by immune response that attackstissues or cells of ones own body, or pathogen-specific immune responsesthat also are harmful for ones own tissues or cells, or non-specificimmune activation which is harmful for ones own tissues or cells.Examples of autoimmune diseases include, but are not limited to,rheumatoid arthritis, SLE, diabetes mellitus, myasthenia gravis,reactive arthritis, ankylosing spondylitis, and multiple sclerosis.These and other inflammatory conditions, including IBD, psoriasis,pancreatitis, and various immunodeficiencies, can be treated usingantigens that are optimized using the methods of the invention.

These conditions are often characterized by an accumulation ofinflammatory cells, such as lymphocytes, macrophages, and neutrophils,at the sites of inflammation. Altered cytokine production levels areoften observed, with increased levels of cytokine production. Severalautoimmune diseases, including diabetes and rheumatoid arthritis, arelinked to certain MHC haplotypes. Other autoimmune-type disorders, suchas reactive arthritis, have been shown to be triggered by bacteria suchas Yersinia and Shigella, and evidence suggests that several otherautoimmune diseases, such as diabetes, multiple sclerosis, rheumatoidarthritis, may also be initiated by viral or bacterial infections ingenetically susceptible individuals.

Current strategies of treatment generally include anti-inflammatorydrugs, such as NSAID or cyclosporin, and antiproliferative drugs, suchas methotrexate. These therapies are non-specific, so a need exists fortherapies having greater specificity, and for means to direct the immuneresponses towards the direction that inhibits the autoimmune process.

The present invention provides several strategies by which these needscan be fulfilled. First, the invention provides methods of obtainingantigens having greater tolerogenicity and/or have improvedantigenicity. In a preferred embodiment, the antigens prepared accordingto the invention exhibit improved induction of tolerance by oraldelivery. Oral tolerance is characterized by induction of immunologicaltolerance after oral administration of large quantities of antigen. Inanimal models, this approach has proven to be a very promising approachto treat autoimmune diseases, and clinical trials are in progress toaddress the efficacy of this approach in the treatment of humanautoimmune diseases, such as rheumatoid arthritis and multiplesclerosis. It has also been suggested that induction of oral toleranceagainst viruses used in gene therapy might reduce the immunogenicity ofgene therapy vectors. However, the amounts of antigen required forinduction of oral tolerance are very high and the methods of theinvention provide a means for obtaining antigens that exhibit asignificant improvement in induction of oral tolerance.

Expression library immunization (Barry et al. (1995) Nature 377: 632) isa particularly useful method of screening for optimal antigens for usein genetic vaccines. For example, to identify autoantigens present inYersinia, Shigella, and the like, one can screen for induction of T cellresponses in HLA-B27 positive individuals. Complexes that includeepitopes of bacterial antigens and MHC molecules associated withautoimmune diseases, e.g., HLA-B27 in association with Yersinia antigenscan be used in the prevention of reactive arthritis and ankylosingspondylitis in HLA-B27 positive individuals.

Screening of optimized antigens can be done in animal models which areknown to those of skill in the art. Examples of suitable models forvarious conditions include collagen induced arthritis, the NFS/sld mousemodel of human Sjogren's syndrome; a 120 kD organ-specific autoantigenrecently identified as an analog of human cytoskeletal protein (α-fodrin(Haneji et al. (1997) Science 276: 604), the New Zealand Black/White F1hybrid mouse model of human SLE, NOD mice, a mouse model of humandiabetes mellitus, fas/fas ligand mutant mice, which spontaneouslydevelop autoimmune and lymphoproliferative disorders (Watanabe-Fukunagaet al. (1992) Nature 356: 314), and experimental autoimmuneencephalomyelitis (EAE), in which myelin basic protein induces a diseasethat resembles human multiple sclerosis.

Autoantigens that can be shuffled according to the methods of theinvention and used in vaccines for treating multiple sclerosis include,but are not limited to, myelin basic protein (Stinissen et al. (1996) J.Neurosci. Res. 45: 500-511) or a fusion protein of myelin basic proteinand proteolipid protein (Elliott et al. (1996) J. Clin. Invest. 98:1602-1612), proteolipid protein (PLP) (Rosener et al. (1997) J.Neuroimmunol. 75: 28-34), 2′,3′-cyclic nucleotide 3′-phosphodiesterase(CNPase) (Rosener et al. (1997) J. Neuroimmunol. 75: 28-34), the EpsteinBarr virus nuclear antigen-1 (EBNA-1) (Vaughan et al. (1996) J.Neuroimmunol. 69: 95-102), HSP70 (Salvetti et al. (1996) J.Neuroimmunol. 65: 143-53; Feldmann et al. (1996) Cell 85: 307).

Target antigens that, after shuffling according to the methods of theinvention, can be used to treat scleroderma, systemic sclerosis, andsystemic lupus erythematosus include, for example, (−2-GPI, 50 kDaglycoprotein (Blank et al. (1994) J. Autoimmun. 7: 441-455), Ku(p70/p80) autoantigen, or its 80-kd subunit protein (Hong et al. (1994)Invest. Ophthalmol. Vis. Sci. 35: 4023-4030; Wang et al. (1994) J. CellSci. 107: 3223-3233), the nuclear autoantigens La (SS-B) and Ro (SS-A)(Huang et al. (1997) J. Clin. Immunol. 17: 212-219; Igarashi et al.(1995) Autoimmunity 22: 33-42; Keech et al. (1996) Clin. Exp. Immunol.104: 255-263; Manoussakis et al. (1995) J. Autoimmun. 8: 959-969; Topferet al. (1995) Proc. Nat'l. Acad. Sci. USA 92: 875-879), proteasome(-type subunit C9 (Feist et al. (1996) J. Exp. Med. 184: 1313-1318),Scleroderma antigens Rpp 30, Rpp 38 or Scl-70 (Eder et al. (1997) Proc.Nat'l. Acad. Sci. USA 94: 1101-1106; Hietarinta et al. (1994) Br. J.Rheumatol. 33: 323-326), the centrosome autoantigen PCM-1 (Bao et al.(1995) Autoimmunity 22: 219-228), polymyositis-scleroderma autoantigen(PM-Scl) (Kho et al. (1997) J. Biol. Chem. 272: 13426-13431),scleroderma (and other systemic autoimmune disease) autoantigen CENP-A(Muro et al. (1996) Clin. Immunol. Immunopathol. 78: 86-89), U5, a smallnuclear ribonucleoprotein (snRNP) (Okano et al. (1996) Clin. Immunol.Immunopathol. 81: 41-47), the 100-kd protein of PM-Scl autoantigen (Geet al. (1996) Arthritis Rheum. 39: 1588-1595), the nucleolar U3- andTh(7-2) ribonucleoproteins (Verheijen et al. (1994) J. Immunol. Methods169: 173-182), the ribosomal protein L7 (Neu et al. (1995) Clin. Exp.Immunol. 100: 198-204), hPop1 (Lygerou et al. (1996) EMBO J. 15:5936-5948), and a 36-kd protein from nuclear matrix antigen (Deng et al.(1996) Arthritis Rheum. 39: 1300-1307).

Hepatic autoimmune disorders can also be treated using improvedrecombinant antigens that are prepared according to the methodsdescribed herein. Among the antigens that are useful in such treatmentsare the cytochromes P450 and UDP-glucuronosyl-transferases(Obermayer-Straub and Manns (1996) Baillieres Clin. Gastroenterol. 10:501-532), the cytochromes P450 2C9 and P450 1A2 (Bourdi et al. (1996)Chem. Res. Toxicol. 9: 1159-1166; Clemente et al. (1997) J. Clin.Endocrinol. Metab. 82: 1353-1361), LC-1 antigen (Klein et al. (1996) J.Pediatr. Gastroenterol. Nutr. 23: 461-465), and a 230-kDaGolgi-associated protein (Funaki et al. (1996) Cell Struct. Funct. 21:63-72).

For treatment of autoimmune disorders of the skin, useful antigensinclude, but are not limited to, the 450 kD human epidermal autoantigen(Fujiwara et al. (1996) J. Invest. Dermatol. 106: 1125-1130), the 230 kDand 180 kD bullous pemphigoid antigens (Hashimoto (1995) Keio J. Med.44: 115-123; Murakami et al. (1996) J. Dermatol. Sci. 13: 112-117),pemphigus foliaceus antigen (desmoglein 1), pemphigus vulgaris antigen(desmoglein 3), BPAg2, BPAg1, and type VII collagen (Batteux et al.(1997) J. Clin. Immunol. 17: 228-233; Hashimoto et al. (1996) JDermatol. Sci. 12: 10-17), a 168-kDa mucosal antigen in a subset ofpatients with cicatricial pemphigoid (Ghohestani et al. (1996) J.Invest. Dermatol. 107: 136-139), and a 218-kd nuclear protein (218-kdMi-2) (Seelig et al. (1995) Arthritis Rheum. 38: 1389-1399).

The methods of the invention are also useful for obtaining improvedantigens for treating insulin dependent diabetes mellitus, using one ormore of antigens which include, but are not limited to, insulin,proinsulin, GAD65 and GAD67, heat-shock protein 65 (hsp65), andislet-cell antigen 69 (ICA69) (French et al. (1997) Diabetes 46: 34-39;Roep (1996) Diabetes 45: 1147-1156; Schloot et al. (1997) Diabetologia40: 332-338), viral proteins homologous to GAD65 (Jones and Crosby(1996) Diabetologia 39: 1318-1324), islet cell antigen-relatedprotein-tyrosine phosphatase (PTP) (Cui et al. (1996) J. Biol. Chem.271: 24817-24823), GM2-1 ganglioside (Cavallo et al. (1996) J.Endocrinol. 0.150: 113-120; Dotta et al. (1996) Diabetes 45: 1193-1196),glutamic acid decarboxylase (GAD) (Nepom (1995) Curr. Opin. Immunol. 7:825-830; Panina-Bordignon et al. (1995) J. Exp. Med. 181: 1923-1927), anislet cell antigen (ICA69) (Karges et al. (1997) Biochim. Biophys. Acta1360: 97-101; Roep et al. (1996) Eur. J. Immunol. 26: 1285-1289), Tep69,the single T cell epitope recognized by T cells from diabetes patients(Karges et al. (1997) Biochim. Biophys. Acta 1360: 97-101), ICA 512, anautoantigen of type I diabetes (Solimena et al. (1996) EMBO J. 15:2102-2114), an islet-cell protein tyrosine phosphatase and the 37-kDaautoantigen derived from it in type I diabetes (including IA-2, IA-2)(La Gasse et al. (1997) Mol. Med. 3: 163-173), the 64 kDa protein fromIn-111 cells or human thyroid follicular cells that isimmunoprecipitated with sera from patients with islet cell surfaceantibodies (ICSA) (Igawa et al. (1996) Endocr. J. 43: 299-306), phogrin,a homologue of the human transmembrane protein tyrosine phosphatase, anautoantigen of type 1 diabetes (Kawasaki et al. (1996) Biochem. Biophys.Res. Commun. 227: 440-447), the 40 kDa and 37 kDa tryptic fragments andtheir precursors IA-2 and IA-2 in IDDM (Lampasona et al. (1996) J.Immunol. 157: 2707-2711; Notkins et al. (1996) J. Autoimmun. 9:677-682), insulin or a cholera toxoid-insulin conjugate (Bergerot et al.(1997) Proc. Nat'l. Acad. Sci. USA 94: 4610-4614), carboxypeptidase H,the human homologue of gp330, which is a renal epithelial glycoproteininvolved in inducing Heymann nephritis in rats, and the 38-kD isletmitochondrial autoantigen (Arden et al. (1996) J. Clin. Invest. 97:551-561.

Rheumatoid arthritis is another condition that is treatable usingoptimized antigens prepared according to the present invention. Usefulantigens for rheumatoid arthritis treatment include, but are not limitedto, the 45 kDa DEK nuclear antigen, in particular onset juvenilerheumatoid arthritis and iridocyclitis (Murray et al. (1997) J.Rheumatol. 24: 560-567), human cartilage glycoprotein-39, an autoantigenin rheumatoid arthritis (Verheijden et al. (1997) Arthritis Rheum. 40:1115-1125), a 68 k autoantigen in rheumatoid arthritis (Blass et al.(1997) Ann. Rheum. Dis. 56: 317-322), collagen (Rosloniec et al. (1995)J. Immunol. 155: 4504-4511), collagen type II (Cook et al. (1996)Arthritis Rheum. 39: 1720-1727; Trentham (1996) Ann. N.Y. Acad. Sci.778: 306-314), cartilage link protein (Guerassimov et al. (1997) J.Rheumatol. 24: 959-964), ezrin, radixin and moesin, which areauto-immune antigens in rheumatoid arthritis (Wagatsuma et al. (1996)Mol. Immunol. 33: 1171-1176), and mycobacterial heat shock protein 65(Ragno et al. (1997) Arthritis Rheum. 40: 277-283).

Also among the conditions for which one can obtain an improved antigensuitable for treatment are autoimmune thyroid disorders. Antigens thatare useful for these applications include, for example, thyroidperoxidase and the thyroid stimulating hormone receptor (Tandon andWeetman (1994) J. R. Coll. Physicians Lond. 28: 10-18), thyroidperoxidase from human Graves' thyroid tissue (Gardas et al. (1997)Biochem. Biophys. Res. Commun. 234: 366-370; Zimmer et al. (1997)Histochem. Cell. Biol. 107: 115-120), a 64-kDa antigen associated withthyroid-associated ophthalmopathy (Zhang et al. (1996) Clin. Immunol.Immunopathol. 80: 236-244), the human TSH receptor (Nicholson et al.(1996) J. Mol. Endocrinol. 16: 159-170), and the 64 kDa protein fromIn-111 cells or human thyroid follicular cells that isimmunoprecipitated with sera from patients with islet cell surfaceantibodies (ICSA) (Igawa et al. (1996) Endocr. J. 43: 299-306).

Other conditions and associated antigens include, but are not limitedto, Sjogren's syndrome (-fodrin; Haneji et al. (1997) Science 276:604-607), myastenia gravis (the human M2 acetylcholine receptor orfragments thereof, specifically the second extracellular loop of thehuman M2 acetylcholine receptor; Fu et al. (1996) Clin. Immunol.Immunopathol. 78: 203-207), vitiligo (tyrosinase; Fishman et al. (1997)Cancer 79: 1461-1464), a 450 kD human epidermal autoantigen recognizedby serum from individual with blistering skin disease, and ulcerativecolitis (chromosomal proteins HMG1 and HMG2; Sobajima et al. (1997)Clin. Exp. Immunol. 107: 135-140).

6. Cancer

Immunotherapy has great promise for the treatment of cancer andprevention of metastasis. By inducing an immune response againstcancerous cells, the body's immune system can be enlisted to reduce oreliminate cancer. Improved antigens obtained using the methods of theinvention provide cancer immunotherapies of increased effectivenesscompared to those that are presently available.

One approach to cancer immunotherapy is vaccination using vaccines thatinclude or encode antigens that are specific for tumor cells or byinjecting the patients with purified recombinant cancer antigens. Themethods of the invention can be used for obtaining antigens that exhibitan enhancement of immune responses against known tumor-specificantigens, and also to search for novel protective antigenic sequences.Antigens having optimized expression, processing, and presentation canbe obtained as described herein. The approach used for each particularcancer can vary. For treatment of hormone-sensitive cancers (forexample, breast cancer and prostate cancer), methods of the inventioncan be used to obtain optimized hormone antagonists. For highlyimmunogenic tumors, including melanoma, one can screen for recombinantantigens that optimally boost the immune response against the tumor.Breast cancer, in contrast, is of relatively low immunogenicity andexhibits slow progression, so individual treatments can be designed foreach patient. Prevention of metastasis is also a goal in design ofcancer vaccines.

Among the tumor-specific antigens that can be used in the antigenshuffling methods of the invention are: bullous pemphigoid antigen 2,prostate mucin antigen (PMA) (Beckett and Wright (1995) Int. J. Cancer62: 703-710), tumor associated Thomsen-Friedenreich antigen (Dahlenborget al. (1997) Int. J. Cancer 70: 63-71), prostate-specific antigen (PSA)(Dannull and Belldegrun (1997) Br. J. Urol. 1: 97-103), luminalepithelial antigen (LEA.135) of breast carcinoma and bladdertransitional cell carcinoma (TCC) (Jones et al. (1997) Anticancer Res.17: 685-687), cancer-associated serum antigen (CASA) and cancer antigen125 (CA 125) (Kierkegaard et al. (1995) Gynecol. Oncol. 59: 251-254),the epithelial glycoprotein 40 (EGP40) (Kievit et al. (1997) Int. J.Cancer 71: 237-245), squamous cell carcinoma antigen (SCC) (Lozza et al.(1997) Anticancer Res. 17: 525-529), cathepsin E (Mota et al. (1997) Am.J. Pathol. 150: 1223-1229), tyrosinase in melanoma (Fishman et al.(1997) Cancer 79: 1461-1464), cell nuclear antigen (PCNA) of cerebralcavemomas (Notelet et al. (1997) Surg. Neurol. 47: 364-370), DF3/MUC1breast cancer antigen (Apostolopoulos et al. (1996) Immunol. Cell. Biol.74: 457-464; Pandey et al. (1995) Cancer Res. 55: 4000-4003),carcinoembryonic antigen (Paone et al. (1996) J Cancer Res. Clin. Oncol.122: 499-503; Schlom et al. (1996) Breast Cancer Res. Treat. 38: 27-39),tumor-associated antigen CA 19-9 (Tolliver and O'Brien (1997) South Med.J. 90: 89-90; Tsuruta et al. (1997) Urol. Int. 58: 20-24), humanmelanoma antigens MART-1/Melan-A27-35 and gp100 (Kawakami and Rosenberg(1997) Int. Rev. Immunol. 14: 173-192; Zajac et al. (1997) Int. J.Cancer 71: 491-496), the T and Tn pancarcinoma (CA) glycopeptideepitopes (Springer (1995) Crit. Rev. Oncog. 6: 57-85), a 35 kDtumor-associated autoantigen in papillary thyroid carcinoma (Lucas etal. (1996) Anticancer Res. 16: 2493-2496), KH-1 adenocarcinoma antigen(Deshpande and Danishefsky (1997) Nature 387: 164-166), the A60mycobacterial antigen (Maes et al. (1996) J Cancer Res. Clin. Oncol.122: 296-300), heat shock proteins (HSPs) (Blachere and Srivastava(1995) Semin. Cancer Biol. 6: 349-355), and MAGE, tyrosinase, melan-Aand gp75 and mutant oncogene products (e.g., p53, ras, and HER-2/neu(Bueler and Mulligan (1996) Mol. Med. 2: 545-555; Lewis and Houghton(1995) Semin. Cancer Biol. 6: 321-327; Theobald et al. (1995) Proc.Nat'l. Acad. Sci. USA 92: 11993-11997).

7. Contraception

Genetic vaccines that contain optimized antigens obtained by the methodsof the invention are also useful for contraception. For example, geneticvaccines can be obtained that encode sperm cell specific antigens, andthus induce anti-sperm immune responses. Vaccination can be achieved by,for example, administration of recombinant bacterial strains, e.g.Salmonella and the like, which express sperm antigen, as well as byinduction of neutralizing anti-hCG antibodies by vaccination by DNAvaccines encoding human chorionic gonadotropin (hCG), or a fragmentthereof.

Sperm antigens which can be used in the genetic vaccines include, forexample, lactate dehydrogenase (LDH-C4), galactosyltransferase (GT),SP-10, rabbit sperm autoantigen (RSA), guinea pig (g)PH-20, cleavagesignal protein (CS-1), HSA-63, human (h)PH-20, and AgX-1 (Zhu and Naz(1994) Arch. Androl. 33: 141-144), the synthetic sperm peptide, P10G(O'Rand et al. (1993) J. Reprod. Immunol. 25: 89-102), the 135 kD, 95kD, 65 kD, 47 kD, 41 kD and 23 kD proteins of sperm, and the FA-1antigen (Naz et al. (1995) Arch. Androl. 35: 225-231), and the 35 kDfragment of cytokeratin 1 (Lucas et al. (1996) Anticancer Res. 16:2493-2496).

The methods of the invention can also be used to obtain genetic vaccinesthat are expressed specifically in testis. For example, polynucleotidesequences that direct expression of genes that are specific to testiscan be used (e.g., fertilization antigen-1 and the like). In addition tosperm antigens, antigens expressed on oocytes or hormones regulatingreproduction may be useful targets of contraceptive vaccines. Forexample, genetic vaccines can be used to generate antibodies againstgonadotropin releasing hormone (GnRH) or zona pellucida proteins (Milleret al. (1997) Vaccine 15:1858-1862). Vaccinations using these moleculeshave been shown to be efficacious in animal models (Miller et al. (1997)Vaccine 15:1858-1862). Another example of a useful component of agenetic contraceptive vaccine is the ovarian zona pellucida glycoproteinZP3 (Tung et al. (1994) Reprod. Fertil. Dev. 6:349-355).

Methods of Selecting and Identifying Optimized Recombinant Antigens

Once one has performed DNA shuffling to obtain a library ofpolynucleotides that encode recombinant antigens, the library issubjected to selection and/or screening to identify those librarymembers that encode antigenic peptides that have improved ability toinduce an immune response to the pathogenic agent. Selection andscreening of recombinant polynucleotides that encode polypeptides havingan improved ability to induce an immune response can involve either invivo and in vitro methods, but most often involves a combination ofthese methods. For example, in a typical embodiment the members of alibrary of recombinant nucleic acids are picked, either individually oras pools. The clones can be subjected to analysis directly, or can beexpressed to produce the corresponding polypeptides. In a presentlypreferred embodiment, an in vitro screen is performed to identify thebest candidate sequences for the in vivo studies. Alternatively, thelibrary can be subjected to in vivo challenge studies directly. Theanalyses can employ either the nucleic acids themselves (e.g., asgenetic vaccines), or the polypeptides encoded by the nucleic acids. Aschematic diagram of a typical strategy is shown in FIG. 5. Both invitro and in vivo methods are described in more detail below.

If a recombination cycle is performed in vitro, the products ofrecombination, i.e., recombinant segments, are sometimes introduced intocells before the screening step. Recombinant segments can also be linkedto an appropriate vector or other regulatory sequences before screening.Alternatively, products of recombination generated in vitro aresometimes packaged in viruses (e.g., bacteriophage) before screening. Ifrecombination is performed in vivo, recombination products can sometimesbe screened in the cells in which recombination occurred. In otherapplications, recombinant segments are extracted from the cells, andoptionally packaged as viruses, before screening.

Often, improvements are achieved after one round of recombination andselection. However, recursive sequence recombination can also beemployed to achieve still further improvements in a desired property, orto bring about new (or “distinct”) properties. Recursive sequencerecombination entails successive cycles of recombination to generatemolecular diversity. That is, one creates a family of nucleic acidmolecules showing some sequence identity to each other but differing inthe presence of mutations. In any given cycle, recombination can occurin vivo or in vitro, intracellularly or extracellularly. Furthermore,diversity resulting from recombination can be augmented in any cycle byapplying prior methods of mutagenesis (e.g., error-prone PCR or cassettemutagenesis) to either the substrates or products for recombination.

In a presently preferred embodiment, polynucleotides that encodeoptimized recombinant antigens are subjected to molecular backcrossing,which provides a means to breed the shuffled chimeras/mutants back to aparental or wild-type sequence, while retaining the mutations that arecritical to the phenotype that provides the optimized immune responses.In addition to removing the neutral mutations, molecular backcrossingcan also be used to characterize which of the many mutations in animproved variant contribute most to the improved phenotype. This cannotbe accomplished in an efficient library fashion by any other method.Backcrossing is performed by shuffling the improved sequence with alarge molar excess of the parental sequences.

The nature of screening or selection depends on what property orcharacteristic is to be acquired or the property or characteristic forwhich improvement is sought, and many examples are discussed below. Itis not usually necessary to understand the molecular basis by whichparticular products of recombination (recombinant segments) haveacquired new or improved properties or characteristics relative to thestarting substrates. For example, a gene that encodes an antigenicpolypeptide can have many component sequences each having a differentintended role (see, e.g., FIG. 4). Each of these component sequences canbe varied and recombined simultaneously. Screening/selection can then beperformed, for example, for recombinant segments that have increasedability to induce an immune response to a pathogenic agent without theneed to attribute such improvement to any of the individual componentsequences of the recombinant polynucleotide.

Depending on the particular screening protocol used for a desiredproperty, initial round(s) of screening can sometimes be performed usingbacterial cells due to high transfection efficiencies and ease ofculture. However, especially for testing of immunogenic activity, testanimals are used for library expression and screening. Similarly othertypes of screening which are not amenable to screening in bacterial orsimple eukaryotic library cells, are performed in cells selected for usein an environment close to that of their intended use. Final rounds ofscreening can be performed in cells or organisms that are as close aspossible to the precise cell type or organism of intended use.

If further improvement in a property is desired, at least one, andusually a collection, of recombinant segments surviving a first round ofscreening/selection are subject to a further round of recombination.These recombinant segments can be recombined with each other or withexogenous segments representing the original substrates or furthervariants thereof. Again, recombination can proceed in vitro or in vivo.If the previous screening step identifies desired recombinant segmentsas components of cells, the components can be subjected to furtherrecombination in vivo, or can be subjected to further recombination invitro, or can be isolated before performing a round of in vitrorecombination. Conversely, if the previous screening step identifiesdesired recombinant segments in naked form or as components of viruses,these segments can be introduced into cells to perform a round of invivo recombination. The second round of recombination, irrespective howperformed, generates further recombinant segments which encompassadditional diversity than is present in recombinant segments resultingfrom previous rounds.

The second round of recombination can be followed by a further round ofscreening/selection according to the principles discussed above for thefirst round. The stringency of screening/selection can be increasedbetween rounds. Also, the nature of the screen and the property beingscreened for can vary between rounds if improvement in more than oneproperty is desired or if acquiring more than one new property isdesired. Additional rounds of recombination and screening can then beperformed until the recombinant segments have sufficiently evolved toacquire the desired new or improved property or function.

The practice of this invention involves the construction of recombinantnucleic acids and the expression of genes in transfected host cells.Molecular cloning techniques to achieve these ends are known in the art.A wide variety of cloning and in vitro amplification methods suitablefor the construction of recombinant nucleic acids such as expressionvectors are well-known to persons of skill. General texts which describemolecular biological techniques useful herein, including mutagenesis,include Berger and Kimmel, Guide to Molecular Cloning Techniques,Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif.(Berger); Sambrook et al., Molecular Cloning—A Laboratory Manual (2ndEd.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,1989 (“Sambrook”) and Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (supplementedthrough 1998) (“Ausubel”)). Examples of techniques sufficient to directpersons of skill through in vitro amplification methods, including thepolymerase chain reaction (PCR) the ligase chain reaction (LCR),Q-replicase amplification and other RNA polymerase mediated techniques(e.g., NASBA) are found in Berger, Sambrook, and Ausubel, as well asMullis et al. (1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide toMethods and Applications (Innis et al. eds) Academic Press Inc. SanDiego, Calif. (1990) (Innis); Arnheim & Levinson (Oct. 1, 1990) C&EN36-47; The Journal Of NIH Research (1991) 3, 81-94; (Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86, 1173; Guatelli et al. (1990) Proc. Natl.Acad. Sci. USA 87, 1874; Lomell et al. (1989) J. Clin. Chem 35, 1826;Landegren et al. (1988) Science 241, 1077-1080; Van Brunt (1990)Biotechnology 8, 291-294; Wu and Wallace (1989) Gene 4, 560; Barringeret al. (1990) Gene 89, 117, and Sooknanan and Malek (1995) Biotechnology13: 563-564. Improved methods of cloning in vitro amplified nucleicacids are described in Wallace et al., U.S. Pat. No. 5,426,039. Improvedmethods of amplifying large nucleic acids by PCR are summarized in Chenget al. (1994) Nature 369: 684-685 and the references therein, in whichPCR amplicons of up to 40 kb are generated. One of skill will appreciatethat essentially any RNA can be converted into a double stranded DNAsuitable for restriction digestion, PCR expansion and sequencing usingreverse transcriptase and a polymerase. See, Ausubel, Sambrook andBerger, all supra.

Oligonucleotides for use as probes, e.g., in in vitro amplificationmethods, for use as gene probes, or as shuffling targets (e.g.,synthetic genes or gene segments) are typically synthesized chemicallyaccording to the solid phase phosphoramidite triester method describedby Beaucage and Caruthers (1981) Tetrahedron Letts., 22(20):1859-1862,e.g., using an automated synthesizer, as described inNeedham-VanDevanter et al. (1984) Nucleic Acids Res., 12:6159-6168.Oligonucleotides can also be custom made and ordered from a variety ofcommercial sources known to persons of skill.

Indeed, essentially any nucleic acid with a known sequence can be customordered from any of a variety of commercial sources, such as The MidlandCertified Reagent Company (mcrc@oligos.com), The Great American GeneCompany (http://www.genco.com), ExpressGen Inc. (wvww.expressgen.com),Operon Technoloigies Inc. (Alameda, Calif.) and many others. Similarly,peptides and antibodies can be custom ordered from any of a variety ofsources, such as PeptidoGenic (pkim@ccnet.com), HTI Bio-products, Inc.(http://www.htibio.com), BMA Biomedicals Ltd (U.K.), Bio'Synthesis,Inc., and many others.

1. Purification and In Vitro Analysis of Recombinant Nucleic Acids andPolypeptides

Once DNA shuffling has been performed, the resulting library ofrecombinant polynucleotides can be subjected to purification andpreliminary analysis in vitro, in order to identify the most promisingcandidate recombinant nucleic acids. Advantageously, the assays can bepracticed in a high-throughput format. For example, to purify individualshuffled recombinant antigens, clones can robotically picked into96-well formats, grown, and, if desired, frozen for storage.

Whole cell lysates (V-antigen), periplasmic extracts, or culturesupernatants (toxins) can be assayed directly by ELISA as describedbelow, but high throughput purification is sometimes also needed.Affinity chromatography using immobilized antibodies or incorporation ofa small nonimmunogenic affinity tag such as a hexahistidine peptide withimmobilized metal affinity chromatography will allow rapid proteinpurification. High binding-capacity reagents with 96-well filter bottomplates provide a high throughput purification process. The scale ofculture and purification will depend on protein yield, but initialstudies will require less than 50 micrograms of protein. Antigensshowing improved properties can be purified in larger scale by FPLC forre-assay and animal challenge studies.

In some embodiments, the shuffled antigen-encoding polynucleotides areassayed as genetic vaccines. Genetic vaccine vectors containing theshuffled antigen sequences can be prepared using robotic colony pickingand subsequent robotic plasmid purification. Robotic plasmidpurification protocols are available that allow purification of 600-800plasmids per day. The quantity and purity of the DNA can also beanalyzed in 96-well plates, for example. In a presently preferredembodiment, the amount of DNA in each sample is robotically normalized,which can significantly reduce the variation between different batchesof vectors.

Once the proteins and/or nucleic acids are picked and purified asdesired, they can be subjected to any of a number of in vitro analysismethods. Such screenings include, for example, phage display, flowcytometry, and ELISA assays to identify antigens that are efficientlyexpressed and have multiple epitopes and a proper folding pattern. Inthe case of bacterial toxins, the libraries may also be screened forreduced toxicity in mammalian cells.

As one example, to identify recombinant antigens that arecross-reactive, one can use a panel of monoclonal antibodies forscreening. A humoral immune response generally targets multiple regionsof antigenic proteins. Accordingly, monoclonal antibodies can be raisedagainst various regions of immunogenic proteins (Alving et al. (1995)Immunol. Rev. 145: 5). In addition, there are several examples ofmonoclonal antibodies that only recognize one strain of a givenpathogen, and by definition, different serotypes of pathogens arerecognized by different sets of antibodies. For example, a panel ofmonoclonal antibodies have been raised against VEE envelope proteins,thus providing a means to recognize different subtypes of the virus(Roehrig and Bolin (1997) J. Clin. Microbiol. 35: 1887). Suchantibodies, combined with phage display and ELISA screening, can be usedto enrich recombinant antigens that have epitopes from multiple pathogenstrains. Flow cytometry based cell sorting will further allow for theselection of variants that are most efficiently expressed.

Phage display provides a powerful method for selecting proteins ofinterest from large libraries (Bass et al. (1990) Proteins: Struct.Funct. Genet. 8: 309; Lowman and Wells (1991) Methods: A Conipanion toMethods Enz. 3(3); 205-216. Lowman and Wells (1993) J. Mol. Biol. 234;564-578). Some recent reviews on the phage display technique include,for example, McGregor (1996) Mol Biotechnol. 6(2):155-62; Dunn (1996)Curr. Opin. Biotechnol. 7(5):547-53; Hill et al. (1996) Mol Microbiol20(4):685-92; Phage Display of Peptides and Proteins: A LaboratoryManual. B K. Kay, J. Winter, J, McCafferty eds., Academic Press 1996;O'Neil et al. (1995) Curr. Opin. Struct. Biol. 5(4):443-9; Phizicky etal. (1995) Microbiol Rev. 59(1):94-123; Clackson et al. (1994) TrendsBiotechnol. 12(5):173-84; Felici et al. (1995) Biotechnol. Annu. Rev.1:149-83; Burton (1995) Immunotechnology 1(2):87-94.) See also, Cwirlaet al., Proc. Natl. Acad. Sci. USA 87: 6378-6382 (1990); Devlin et al.,Science 249: 404-406 (1990), Scott & Smith, Science 249: 386-388 (1990);Ladner et al., U.S. Pat. No. 5,571,698. Each phage particle displays aunique variant protein on its surface and packages the gene encodingthat particular variant. The shuffled genes for the antigens are fusedto a protein that is expressed on the phage surface, e.g., gene III ofphage M13, and cloned into phagemid vectors. In a presently preferredembodiment, a suppressible stop codon (e.g., an amber stop codon)separates the genes so that in a suppressing strain of E. coli, theantigen-gIIIp fusion is produced and becomes incorporated into phageparticles upon infection with M13 helper phage. The same vector candirect production of the unfused antigen alone in a nonsuppressing E.coli for protein purification.

The genetic packages most frequently used for display libraries arebacteriophage, particularly filamentous phage, and especially phage M13,Fd and F1. Most work has involved inserting libraries encodingpolypeptides to be displayed into either gIII or gVIII of these phageforming a fusion protein. See, e.g., Dower, WO 91/19818; Devlin, WO91/18989; MacCafferty, WO 92/01047 (gene III); Huse, WO 92/06204; Kang,WO 92/18619 (gene VIII). Such a fusion protein comprises a signalsequence, usually but not necessarily, from the phage coat protein, apolypeptide to be displayed and either the gene III or gene VIII proteinor a fragment thereof. Exogenous coding sequences are often inserted ator near the N-terminus of gene III or gene VIII although other insertionsites are possible.

Eukaryotic viruses can be used to display polypeptides in an analogousmanner. For example, display of human heregulin fused to gp70 of Moloneymurine leukemia virus has been reported by Han et al., Proc. Natl. Acad.Sci. USA 92: 9747-9751 (1995). Spores can also be used as replicablegenetic packages. In this case, polypeptides are displayed from theouter surface of the spore. For example, spores from B. subtilis havebeen reported to be suitable. Sequences of coat proteins of these sporesare provided by Donovan et al., J. Mol. Biol. 196, 1-10 (1987). Cellscan also be used as replicable genetic packages. Polypeptides to bedisplayed are inserted into a gene encoding a cell protein that isexpressed on the cells surface. Bacterial cells including Salmonellatyphimurium, Bacillus subtilis, Pseudomonas aeruginosa, Vibrio cholerae,Klebsiella pneumonia, Neisseria gonorrhoeae, Neisseria meningitidis,Bacteroides nodosus, Moravella bovis, and especially Escherichia coliare preferred. Details of outer surface proteins are discussed by Ladneret al., U.S. Pat. No. 5,571,698 and references cited therein. Forexample, the lamB protein of E. coli is suitable.

A basic concept of display methods that use phage or other replicablegenetic package is the establishment of a physical association betweenDNA encoding a polypeptide to be screened and the polypeptide. Thisphysical association is provided by the replicable genetic package,which displays a polypeptide as part of a capsid enclosing the genome ofthe phage or other package, wherein the polypeptide is encoded by thegenome. The establishment of a physical association between polypeptidesand their genetic material allows simultaneous mass screening of verylarge numbers of phage bearing different polypeptides. Phage displayinga polypeptide with affinity to a target, e.g., a receptor, bind to thetarget and these phage are enriched by affinity screening to the target.The identity of polypeptides displayed from these phage can bedetermined from their respective genomes. Using these methods apolypeptide identified as having a binding affinity for a desired targetcan then be synthesized in bulk by conventional means, or thepolynucleotide that encodes the peptide or polypeptide can be used aspart of a genetic vaccine.

Variants with specific binding properties, in this case binding tofamily-specific antibodies, are easily enriched by panning withimmobilized antibodies. Antibodies specific for a single family are usedin each round of panning to rapidly select variants that have multipleepitopes from the antigen families. For example, A-family specificantibodies can be used to select those shuffled clones that displayA-specific epitopes in the first round of panning. A second round ofpanning with B-specific antibodies will select from the “A” clones thosethat display both A- and B-specific epitopes. A third round of panningwith C-specific antibodies will select for variants with A, B, and Cepitopes. A continual selection exists during this process for clonesthat express well in E. coli and that are stable throughout theselection. Improvements in factors such as transcription, translation,secretion, folding and stability are often observed and will enhance theutility of selected clones for use in vaccine production.

Phage ELISA methods can be used to rapidly characterize individualvariants. These assays provide a rapid method for quantitation ofvariants without requiring purification of each protein. Individualclones are arrayed into 96-well plates, grown, and frozen for storage.Cells in duplicate plates are infected with helper phage, grownovernight and pelleted by centrifugation. The supernatants containingphage displaying particular variants are incubated with immobilizedantibodies and bound clones are detected by anti-M13 antibodyconjugates. Titration series of phage particles, immobilized antigen,and/or soluble antigen competition binding studies are all highlyeffective means to quantitate protein binding. Variant antigensdisplaying multiple epitopes will be further studied in appropriateanimal challenge models.

Several groups have reported an in vitro ribosome display system for thescreening and selection of mutant proteins with desired properties fromlarge libraries. This technique can be used similarly to phage displayto select or enrich for variant antigens with improved properties suchas broad cross reactivity to antibodies and improved folding (see, e.g.,Hanes et al. (1997) Proc. Nat'l. Acad. Sci. USA 94(10):4937-42;Mattheakis et al. (1994) Proc. Nat'l. Acad. Sci. USA 91(19):9022-6; Heet al. (1997) Nucl. Acids Res. 25(24):5132-4; Nemoto et al. (1997) FEBSLett. 414(2):405-8).

Other display methods exist to screen antigens for improved propertiessuch as increased expression levels, broad cross reactivity, enhancedfolding and stability. These include, but are not limited to display ofproteins on intact E. coli or other cells. (e.g., Francisco et al.(1993) Proc. Nat'l. Acad. Sci. USA 90: 1044-10448; Lu et al. (1995)Bio/Technology 13: 366-372). Fusions of shuffled antigens to DNA-bindingproteins can link the antigen protein to its gene in an expressionvector (Schatz et al. (1996) Methods Enzymol. 267: 171-91; Gates et al.(1996) J. Mol. Biol. 255: 373-86.)

The various display methods and ELISA assays can be used to screen forshuffled antigens with improved properties such as presentation ofmultiple epitopes, improved immunogenicity, increased expression levels,increased folding rates and efficiency, increased stability to factorssuch as temperature, buffers, solvents, improved purificationproperties, etc. Selection of shuffled antigens with improvedexpression, folding, stability and purification profile under a varietyof chromatographic conditions can be very important improvements toincorporate for the vaccine manufacturing process.

To identify recombinant antigenic polypeptides that exhibit improvedexpression in a host cell, flow cytometry is a useful technique. Flowcytometry provides a method to efficiently analyze the functionalproperties of millions of individual cells. One can analyze theexpression levels of several genes simultaneously, and flowcytometry-based cell sorting allows for the selection of cells thatdisplay properly expressed antigen variants on the cell surface or inthe cytoplasm. Very large numbers (>10⁷) of cells can be evaluated in asingle vial experiment, and the pool of the best individual sequencescan be recovered from the sorted cells. These methods are particularlyuseful in the case of, for example, Hantaan virus glycoproteins, whichare generally very poorly expressed in mammalian cells. This approachprovides a general solution to improve expression levels of pathogenantigens in mammalian cells, a phenomenon that is critical for thefunction of genetic vaccines.

To use flow cytometry to analyze polypeptides that are not expressed onthe cell surface, one can engineer the recombinant polynucleotides inthe library such that the polynucleotide is expressed as a fusionprotein that has a region of amino acids which is targeted to the cellmembrane. For example, the region can encode a hydrophobic stretch ofC-terminal amino acids which signals the attachment of aphosphoinositol-glycan (PIG) terminus on the expressed protein anddirects the protein to be expressed on the surface of the transfectedcell (Whitehorn et al. (1995) Biotechnology (N Y) 13:1215-9). With anantigen that is naturally a soluble protein, this method will likely notaffect the three dimensional folding of the protein in this engineeredfusion with a new C-terminus. With an antigen that is naturally atransmembrane protein (e.g., a surface membrane protein on pathogenicviruses, bacteria, protozoa or tumor cells) there are at least twopossibilities. First, the extracellular domain can be engineered to bein fusion with the C-terminal sequence for signaling PIG-linkage.Second, the protein can be expressed in toto relying on the signallingof the host cell to direct it efficiently to the cell surface. In aminority of cases, the antigen for expression will have an endogenousPIG terminal linkage (e.g., some antigens of pathogenic protozoa).

Those cells expressing the antigen can be identified with a fluorescentmonoclonal antibody specific for the C-terminal sequence on PIG-linkedforms of the surface antigen. FACS analysis allows quantitativeassessment of the level of expression of the correct form of the antigenon the cell population. Cells expressing the maximal level of antigenare sorted and standard molecular biology methods are used to recoverthe plasmid DNA vaccine vector that conferred this reactivity. Analternative procedure that allows purification of all those cellsexpressing the antigen (and that may be useful prior to loading onto acell sorter since antigen expressing cells may be a very small minoritypopulation), is to rosette or pan-purify the cells expressing surfaceantigen. Rosettes can be formed between antigen expressing cells anderythrocytes bearing covalently coupled antibody to the relevantantigen. These are readily purified by unit gravity sedimentation.Panning of the cell population over petri dishes bearing immobilizedmonoclonal antibody specific for the relevant antigen can also be usedto remove unwanted cells.

In the high throughput assays of the invention, it is possible to screenup to several thousand different shuffled variants in a single day. Forexample, each well of a microtiter plate can be used to run a separateassay, or, if concentration or incubation time effects are to beobserved, every 5-10 wells can test a single variant. Thus, a singlestandard microtiter plate can assay about 100 (e.g., 96) reactions. If1536 well plates are used, then a single plate can easily assay fromabout 100 to about 1500 different reactions. It is possible to assayseveral different plates per day; assay screens for up to about6,000-20,000 different assays (i.e., involving different nucleic acids,encoded proteins, concentrations, etc.) is possible using the integratedsystems of the invention. More recently, microfluidic approaches toreagent manipulation have been developed, e.g., by Caliper Technologies(Palo Alto, Calif.).

In one aspect, library members, e.g., cells, viral plaques, or the like,are separated on solid media to produce individual colonies (orplaques). Using an automated colony picker (e.g., the Q-bot, Genetix,U.K.), colonies or plaques are identified, picked, and up to 10,000different mutants inoculated into 96 well microtiter dishes, optionallycontaining glass balls in the wells to prevent aggregation. The Q-botdoes not pick an entire colony but rather inserts a pin through thecenter of the colony and exits with a small sampling of cells (orviruses in plaque applications). The time the pin is in the colony, thenumber of dips to inoculate the culture medium, and the time the pin isin that medium each effect inoculum size, and each can be controlled andoptimized. The uniform process of the Q-bot decreases human handlingerror and increases the rate of establishing cultures (roughly 10,000/4hours). These cultures are then shaken in a temperature and humiditycontrolled incubator. The glass balls in the microtiter plates act topromote uniform aeration of cells dispersal of cells, or the like,similar to the blades of a fermentor. Clones from cultures of interestcan be cloned by limiting dilution. Plaques or cells constitutinglibraries can also be screened directly for production of proteins,either by detecting hybridization, protein activity, protein binding toantibodies, or the like.

The ability to detect a subtle increase in the performance of a shuffledlibrary member over that of a parent strain relies on the sensitivity ofthe assay. The chance of finding the organisms having an improvement inability to induce an immune response is increased by the number ofindividual mutants that can be screened by the assay. To increase thechances of identifying a pool of sufficient size, a prescreen thatincreases the number of mutants processed by 10-fold can be used. Thegoal of the prescreen will be to quickly identify mutants having equalor better product titers than the parent strain(s) and to move onlythese mutants forward to liquid cell culture for subsequent analysis.

A number of well known robotic systems have also been developed forsolution phase chemistries useful in assay systems. These systemsinclude automated workstations like the automated synthesis apparatusdeveloped by Takeda Chemical Industries, LTD. (Osaka, Japan) and manyrobotic systems utilizing robotic arms (Zymate II, Zymark Corporation,Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.) which mimicthe manual synthetic operations performed by a scientist. Any of theabove devices are suitable for use with the present invention, e.g., forhigh-throughput screening of molecules encoded by codon-altered nucleicacids. The nature and implementation of modifications to these devices(if any) so that they can operate as discussed herein with reference tothe integrated system will be apparent to persons skilled in therelevant art.

High throughput screening systems are commercially available (see, e.g.,Zymark Corp., Hopkinton, Mass.; Air Technical Industries, Mentor, Ohio;Beckman Instruments, Inc. Fullerton, Calif.; Precision Systems, Inc.,Natick, Mass., etc.). These systems typically automate entire proceduresincluding all sample and reagent pipetting, liquid dispensing, timedincubations, and final readings of the microplate in detector(s)appropriate for the assay. These configurable systems provide highthroughput and rapid start up as well as a high degree of flexibilityand customization.

The manufacturers of such systems provide detailed protocols the varioushigh throughput. Thus, for example, Zymark Corp. provides technicalbulletins describing screening systems for detecting the modulation ofgene transcription, ligand binding, and the like. Microfluidicapproaches to reagent manipulation have also been developed, e.g., byCaliper Technologies (Palo Alto, Calif.).

Optical images viewed (and, optionally, recorded) by a camera or otherrecording device (e.g., a photodiode and data storage device) areoptionally further processed in any of the embodiments herein, e.g., bydigitizing the image and/or storing and analyzing the image on acomputer. As noted above, in some applications, the signals resultingfrom assays are florescent, making optical detection approachesappropriate in these instances. A variety of commercially availableperipheral equipment and software is available for digitizing, storingand analyzing a digitized video or digitized optical image, e.g., usingPC (Intel x86 or Pentium chip-compatible DOS, OS2 WINDOWS, WINDOWS NT orWINDOWS95 based machines), MACINTOSH, or UNIX based (e.g., SUN workstation) computers.

One conventional system carries light from the assay device to a cooledcharge-coupled device (CCD) camera, in common use in the art. A CCDcamera includes an array of picture elements (pixels). The light fromthe specimen is imaged on the CCD. Particular pixels corresponding toregions of the specimen (e.g., individual hybridization sites on anarray of biological polymers) are sampled to obtain light intensityreadings for each position. Multiple pixels are processed in parallel toincrease speed. The apparatus and methods of the invention are easilyused for viewing any sample, e.g., by fluorescent or dark fieldmicroscopic techniques.

Integrated systems for analysis in the present invention typicallyinclude a digital computer with high-throughput liquid control software,image analysis software, data interpretation software, a robotic liquidcontrol armature for transferring solutions from a source to adestination operably linked to the digital computer, an input device(e.g., a computer keyboard) for entering data to the digital computer tocontrol high throughput liquid transfer by the robotic liquid controlarmature and, optionally, an image scanner for digitizing label signalsfrom labeled assay component. The image scanner interfaces with theimage analysis software to provide a measurement of optical intensity.Typically, the intensity measurement is interpreted by the datainterpretation software to show whether the optimized recombinantantigenic polypeptide products are produced.

2. Antigen Library Immunization

In a presently preferred embodiment, antigen library immunization (ALI)is used to identify optimized recombinant antigens that have improvedimmunogenicity. ALI involves introduction of the library of recombinantantigen-encoding nucleic acids, or the recombinant antigens encoded bythe shuffled nucleic acids, into a test animal. The animals are thensubjected to in vivo challenge using live pathogens. Neutralizingantibodies and cross-protective immune responses are studied afterimmunization with the entire libraries, pools and/or individual antigenvariants.

Methods of immunizing test animals are well known to those of skill inthe art. In presently preferred embodiments, test animals are immunizedtwice or three times at two week intervals. One week after the lastimmunization, the animals are challenged with live pathogens (ormixtures of pathogens), and the survival and symptoms of the animals isfollowed. Immunizations using test animal challenge are described in,for example, Roggenkamp et al. (1997) Infect. Immun. 65: 446; Woody etal. (1997) Vaccine 2: 133; Agren et al. (1997) J. Immunol. 158: 3936;Konishi et al. (1992) Virology 190: 454; Kinney et al. (1988) J. Virol.62: 4697; Iacono-Connors et al. (1996) Virus Res. 43: 125; Kochel et al.(1997) Vaccine 15: 547; and Chu et al. (1995) J. Virol. 69: 6417.

The immunizations can be performed by injecting either the recombinantpolynucleotides themselves, i.e., as a genetic vaccine, or by immunizingthe animals with polypeptides encoded by the recombinantpolynucleotides. Bacterial antigens are typically screened primarily asrecombinant proteins, whereas viral antigens are preferably analyzedusing genetic vaccinations.

To dramatically reduce the number of experiments required to identifyindividual antigens having improved immunogenic properties, one can usepooling and deconvolution, as diagrammed in FIG. 6. Pools of recombinantnucleic acids, or polypeptides encoded by the recombinant nucleic acids,are used to immunize test animals. Those pools that result in protectionagainst pathogen challenge are then subdivided and subjected toadditional analysis. The high throughput in vitro approaches describedabove can be used to identify the best candidate sequences for the invivo studies.

The challenge models that can be used to screen for protective antigensinclude pathogen and toxin models, such as Yersinia bacteria, bacterialtoxins (such as Staphylococcal and Streptococcal enterotoxins, E.coli/V. cholerae enterotoxins), Venezuelan equine encephalitis virus(VEE), Flaviviruses (Japanese encephalitis virus, Tick-borneencephalitis virus, Dengue virus), Hantaan virus, Herpes simplex,influenza virus (e.g., Influenza A virus), Vesicular Stomatitis Virus,Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli,Klebsiella pneumoniae, Toxoplasma gondii, Plasmodium yoelii, Herpessimplex, influenza virus (e.g., Influenza A virus), and VesicularStomatitis Virus. However, the test animals can also be challenged withtumor cells to enable screening of antigens that efficiently protectagainst malignancies. Individual shuffled antigens or pools of antigensare introduced into the animals intradermally, intramuscularly,intravenously, intratracheally, anally, vaginally, orally, orintraperitoneally and antigens that can prevent the disease are chosen,when desired, for further rounds of shuffling and selection. Eventually,the most potent antigens, based on in vivo data in test animals andcomparative in vitro studies in animals and man, are chosen for humantrials, and their capacity to prevent and treat human diseases isinvestigated.

In some embodiments, antigen library immunization and pooling ofindividual clones is used to immunize against a pathogen strain that wasnot included in the sequences that were used to generate the library.The level of crossprotection provided by different strains of a givenpathogen can significantly. However, homologous titer is always higherthan heterologous titer. Pooling and deconvolution is especiallyefficient in models where minimal protection is provided by thewild-type antigens used as starting material for shuffling (for exampleminimal protection by antigens A and B against strain C in FIG. 3B).This approach can be taken, for example, when evolving the V-antigen ofYersinae or Hantaan virus glycoproteins.

In some embodiments, the desired screening involves analysis of theimmune response based on immunological assays known to those skilled inthe art. Typically, the test animals are first immunized and blood ortissue samples are collected for example one to two weeks after the lastimmunization. These studies enable one to one can measure immuneparameters that correlate to protective immunity, such as induction ofspecific antibodies (particularly IgG) and induction of specific Tlymphocyte responses, in addition to determining whether an antigen orpools of antigens provides protective immunity. Spleen cells orperipheral blood mononuclear cells can be isolated from immunized testanimals and measured for the presence of antigen-specific T cells andinduction of cytokine synthesis. ELISA, ELISPOT and cytoplasmic cytokinestaining, combined with flow cytometry, can provide such information ona single-cell level.

Common immunological tests that can be used to identify the efficacy ofimmunization include antibody measurements, neutralization assays andanalysis of activation levels or frequencies of antigen presenting cellsor lymphocytes that are specific for the antigen or pathogen. The testanimals that can be used in such studies include, but are not limitedto, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, pigs andmonkeys. Monkey is a particularly useful test animal because the MHCmolecules of monkeys and humans are very similar.

Virus neutralization assays are useful for detection of antibodies thatnot only specifically bind to the pathogen, but also neutralize thefunction of the virus. These assays are typically based on detection ofantibodies in the sera of immunized animal and analysis of theseantibodies for their capacity to inhibit viral growth in tissue culturecells. Such assays are known to those skilled in the art. One example ofa virus neutralization assay is described by Dolin R (J. Infect. Dis.1995, 172:1175-83). Virus neutralization assays provide means to screenfor antigens that also provide protective immunity.

In some embodiments, shuffled antigens are screened for their capacityto induce T cell activation in vivo. More specifically, peripheral bloodmononuclear cells or spleen cells from injected mice can be isolated andthe capacity of cytotoxic T lymphocytes to lyse infected, autologoustarget cells is studied. The spleen cells can be reactivated with thespecific antigen in vitro. In addition, T helper cell activation anddifferentiation is analyzed by measuring cell proliferation orproduction of T_(H)1 (IL-2 and IFN-γ) and T_(H)2 (IL-4 and IL-5)cytokines by ELISA and directly in CD4⁺ T cells by cytoplasmic cytokinestaining and flow cytometry. Based on the cytokine production profile,one can also screen for alterations in the capacity of the antigens todirect T_(H)1/T_(H)2 differentiation (as evidenced, for example, bychanges in ratios of IL-4/IFN-γ, IL-4/IL-2, IL-5/IFN-γ, IL-5/IL-2,IL-13/IFN-γ, IL-13/IL-2). The analysis of the T cell activation inducedby the antigen variants is a very useful screening method, becausepotent activation of specific T cells in vivo correlates to induction ofprotective immunity.

The frequency of antigen-specific CD8⁺ T cells in vivo can also bedirectly analyzed using tetramers of MHC class I molecules expressingspecific peptides derived from the corresponding pathogen antigens (Oggand McMichael, Curr. Opin. Immunol. 1998, 10:393-6; Altman et al.,Science 1996, 274:94-6). The binding of the tetramers can be detectedusing flow cytometry, and will provide information about the efficacy ofthe shuffled antigens to induce activation of specific T cells. Forexample, flow cytometry and tetramer stainings provide an efficientmethod of identifying T cells that are specific to a given antigen orpeptide. Another method involves panning using plates coated withtetramers with the specific peptides. This method allows large numbersof cells to be handled in a short time, but the method only selects forhighest expression levels. The higher the frequency of antigen-specificT cells in vivo is, the more efficient the immunization has been,enabling identification of the antigen variants that have the mostpotent capacity to induce protective immune responses. These studies areparticularly useful when conducted in monkeys, or other primates,because the MHC class I molecules of humans mimic those of otherprimates more closely than those of mice.

Measurement of the activation of antigen presenting cells (APC) inresponse to immunization by antigen variants is another useful screeningmethod. Induction of APC activation can be detected based on changes insurface expression levels of activation antigens, such as B7-1 (CD80),B7-2 (CD86), MHC class I and II, CD14, CD23, and Fc receptors, and thelike.

Shuffled cancer antigens that induce cytotoxic T cells that have thecapacity to kill cancer cells can be identified by measuring thecapacity of T cells derived from immunized animals to kill cancer cellsin vitro. Typically the cancer cells are first labeled with radioactiveisotopes and the release of radioactivity is an indication of tumor cellkilling after incubation in the presence of T cells from immunizedanimals. Such cytotoxicity assays are known in the art.

An indication of the efficacy of an antigen to activate T cells specificfor, for example, cancer antigens, allergens or autoantigens, is alsothe degree of skin inflammation when the antigen is injected into theskin of a patient or test animal. Strong inflammation is correlated withstrong activation of antigen-specific T cells. Improved activation oftumor-specific T cells may lead to enhanced killing of the tumors. Incase of autoantigens, one can add immunomodulators that skew theresponses towards T_(H) ², whereas in the case of allergens a T_(H)1response is desired. Skin biopsies can be taken, enabling detailedstudies of the type of immune response that occurs at the sites of eachinjection (in mice and monkeys large numbers of injections/antigens canbe analyzed). Such studies include detection of changes in expression ofcytokines, chemokines, accessory molecules, and the like, by cells uponinjection of the antigen into the skin.

To screen for antigens that have optimal capacity to activateantigen-specific T cells, peripheral blood mononuclear cells frompreviously infected or immunized humans individuals can be used. This isa particularly useful method, because the MHC molecules that willpresent the antigenic peptides are human MHC molecules. Peripheral bloodmononuclear cells or purified professional antigen-presenting cells(APCs) can be isolated from previously vaccinated or infectedindividuals or from patients with acute infection with the pathogen ofinterest. Because these individuals have increased frequencies ofpathogen-specific T cells in circulation, antigens expressed in PBMCs orpurified APCs of these individuals will induce proliferation andcytokine production by antigen-specific CD4⁺ and CD8⁺ T cells. Thus,antigens that simultaneously harbor epitopes from several antigens canbe recognized by their capacity to stimulate T cells from variouspatients infected or immunized with different pathogen antigens, cancerantigens, autoantigens or allergens. One buffy coat derived from a blooddonor contains lymphocytes from 0.5 liters of blood, and up to 10⁴ PBMCcan be obtained, enabling very large screening experiments using T cellsfrom one donor.

When healthy vaccinated individuals (lab volunteers) are studied, onecan make EBV-transformed B cell lines from these individuals. These celllines can be used as antigen presenting cells in subsequent experimentsusing blood from the same donor; this reduces interassay anddonor-to-donor variation. In addition, one can make antigen-specific Tcell clones, after which antigen variants are introduced to EBVtransformed B cells. The efficiency with which the transformed B cellsinduce proliferation of the specific T cell clones is then studied. Whenworking with specific T cell clones, the proliferation and cytokinesynthesis responses are significantly higher than when using totalPBMCs, because the frequency of antigen-specific T cells among PBMC isvery low.

CTL epitopes can be presented by most cells types since the class Imajor histocompatibility complex (MHC) surface glycoproteins are widelyexpressed. Therefore, transfection of cells in culture by libraries ofshuffled antigen sequences in appropriate expression vectors can lead toclass I epitope presentation. If specific CTLs directed to a givenepitope have been isolated from an individual, then the co-culture ofthe transfected presenting cells and the CTLs can lead to release by theCTLs of cytokines, such as IL-2, IFN-γ, or TNF, if the epitope ispresented. Higher amounts of released TNF will correspond to moreefficient processing and presentation of the class I epitope from theshuffled, evolved sequence. Shuffled antigens that induce cytotoxic Tcells that have the capacity to kill infected cells can also beidentified by measuring the capacity of T cells derived from immunizedanimals to kill infected cells in vitro. Typically the target cells arefirst labeled with radioactive isotopes and the release of radioactivityis an indication of target cell killing after incubation in the presenceof T cells from immunized animals. Such cytotoxicity assays are known inthe art.

A second method for identifying optimized CTL epitopes does not requirethe isolation of CTLs reacting with the epitope. In this approach, cellsexpressing class I MHC surface glycoproteins are transfected with thelibrary of evolved sequences as above. After suitable incubation toallow for processing and presentation, a detergent soluble extract isprepared from each cell culture and after a partial purification of theMHC-epitope complex (perhaps optional) the products are submitted tomass spectrometry (Henderson et al. (1993) Proc. Nat'l. Acad. Sci. USA90: 10275-10279). Since the sequence is known of the epitope whosepresentation to be increased, one can calibrate the mass spectrogram toidentify this peptide. In addition, a cellular protein can be used forinternal calibration to obtain a quantitative result; the cellularprotein used for internal calibration could be the MHC molecule itself.Thus one can measure the amount of peptide epitope bound as a proportionof the MHC molecules.

Use of Recombinant Multivalent Antigens

The multivalent antigens of the invention are useful for treating and/orpreventing the various diseases and conditions with which the respectiveantigens are associated. For example, the multivalent antigens can beexpressed in a suitable host cell and are administered in polypeptideform. Suitable formulations and dosage regimes for vaccine delivery arewell known to those of skill in the art.

In presently preferred embodiments, the optimized recombinantpolynucleotides that encode improved allergens are used in conjunctionwith a genetic vaccine vector. The choice of vector and components canalso be optimized for the particular purpose of treating allergy, forexample, or other conditions. For example, the polynucleotide thatencodes the recombinant antigenic polypeptide can be placed under thecontrol of a promoter, e.g., a high activity or tissue-specificpromoter. The promoter used to express the antigenic polypeptide canitself be optimized using recombination and selection methods analogousto those described herein. The vector can contain immunostimulatorysequences such as are described in copending, commonly assigned U.S.patent application Ser. No. ______, entitled “Optimization ofImmunomodulatory Molecules,” filed as TTC Attorney Docket No.18097-030300US on Feb. 10, 1999. A vector engineered to direct a T_(H)1response is preferred for many of the immune responses mediated by theantigens described herein (see, e.g., copending, commonly assigned U.S.patent application Ser. No. ______, entitled “Genetic Vaccine VectorEngineering,” filed on Feb. 10, 1999 as TTC Attorney Docket No.18097-030100US). It is sometimes advantageous to employ a geneticvaccine that is targeted for a particular target cell type (e.g., anantigen presenting cell or an antigen processing cell); suitabletargeting methods are described in copending, commonly assigned U.S.patent application Ser. No. ______, entitled “Targeting of GeneticVaccine Vectors,” filed on Feb. 10, 1999 as TTC Attorney Docket No.18097-030200US.

Genetic vaccines that encode the multivalent antigens described hereincan be delivered to a mammal (including humans) to induce a therapeuticor prophylactic immune response. Vaccine delivery vehicles can bedelivered in vivo by administration to an individual patient, typicallyby systemic administration (e.g., intravenous, intraperitoneal,intramuscular, subdermal, intracranial, anal, vaginal, oral, buccalroute or they can be inhaled) or they can be administered by topicalapplication. Alternatively, vectors can be delivered to cells ex vivo,such as cells explanted from an individual patient (e.g., lymphocytes,bone marrow aspirates, tissue biopsy) or universal donor hematopoieticstem cells, followed by reimplantation of the cells into a patient,usually after selection for cells which have incorporated the vector.

A large number of delivery methods are well known to those of skill inthe art. Such methods include, for example liposome-based gene delivery(Debs and Zhu (1993) WO 93/24640; Mannino and Gould-Fogerite (1988)BioTechniques 6(7): 682-691; Rose U.S. Pat. No. 5,279,833; Brigham(1991) WO 91/06309; and Felgner et al. (1987) Proc. Natl. Acad. Sci. USA84: 7413-7414), as well as use of viral vectors (e.g., adenoviral (see,e.g., Berns et al. (1995) Ann. NY Acad. Sci. 772: 95-104; Ali et al.(1994) Gene Ther. 1: 367-384; and Haddada et al. (1995) Curr. Top.Microbiol. Immunol. 199 (Pt 3): 297-306 for review), papillomaviral,retroviral (see, e.g., Buchscher et al. (1992) J. Virol. 66(5)2731-2739; Johann et al. (1992) J. Virol. 66 (5):1635-1640 (1992);Sommerfelt et al., (1990) Virol. 176:58-59; Wilson et al. (1989) J.Virol. 63:2374-2378; Miller et al., J. Virol. 65:2220-2224 (1991);Wong-Staal et al., PCT/US94/05700, and Rosenburg and Fauci (1993) inFundamental Immunology, Third Edition Paul (ed) Raven Press, Ltd., NewYork and the references therein, and Yu et al., Gene Therapy (1994)supra.), and adeno-associated viral vectors (see, West et al. (1987)Virology 160:38-47; Carter et al. (1989) U.S. Pat. No. 4,797,368; Carteret al. WO 93/24641 (1993); Kotin (1994) Human Gene Therapy 5:793-801;Muzyczka (1994) J. Clin. Invst. 94:1351 and Samulski (supra) for anoverview of AAV vectors; see also, Lebkowski, U.S. Pat. No. 5,173,414;Tratschin et al. (1985) Mol. Cell. Biol. 5(11):3251-3260; Tratschin, etal. (1984) Mol. Cell. Biol., 4:2072-2081; Hermonat and Muzyczka (1984)Proc. Natl. Acad. Sci. USA, 81:6466-6470; McLaughlin et al. (1988) andSamulski et al. (1989) J. Virol., 63:03822-3828), and the like.

“Naked” DNA and/or RNA that comprises a genetic vaccine can beintroduced directly into a tissue, such as muscle. See, e.g., U.S. Pat.No. 5,580,859. Other methods such as “biolistic” or particle-mediatedtransformation (see, e.g., Sanford et al., U.S. Pat. No. 4,945,050; U.S.Pat. No. 5,036,006) are also suitable for introduction of geneticvaccines into cells of a mammal according to the invention. Thesemethods are useful not only for in vivo introduction of DNA into amammal, but also for ex vivo modification of cells for reintroductioninto a mammal. As for other methods of delivering genetic vaccines, ifnecessary, vaccine administration is repeated in order to maintain thedesired level of immunomodulation.

Genetic vaccine vectors (e.g., adenoviruses, liposomes,papillomaviruses, retroviruses, etc.) can be administered directly tothe mammal for transduction of cells in vivo. The genetic vaccinesobtained using the methods of the invention can be formulated aspharmaceutical compositions for administration in any suitable manner,including parenteral (e.g., subcutaneous, intramuscular, intradermal, orintravenous), topical, oral, rectal, intrathecal, buccal (e.g.,sublingual), or local administration, such as by aerosol ortransdermally, for prophylactic and/or therapeutic treatment.Pretreatment of skin, for example, by use of hair-removing agents, maybe useful in transdermal delivery. Suitable methods of administeringsuch packaged nucleic acids are available and well known to those ofskill in the art, and, although more than one route can be used toadminister a particular composition, a particular route can oftenprovide a more immediate and more effective reaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention. A variety of aqueous carriers can be used, e.g.,buffered saline and the like. These solutions are sterile and generallyfree of undesirable matter. These compositions may be sterilized byconventional, well known sterilization techniques. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, for example, sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate and the like. The concentration of genetic vaccine vector inthese formulations can vary widely, and will be selected primarily basedon fluid volumes, viscosities, body weight and the like in accordancewith the particular mode of administration selected and the patient'sneeds.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the packaged nucleic acidsuspended in diluents, such as water, saline or PEG 400; (b) capsules,sachets or tablets, each containing a predetermined amount of the activeingredient, as liquids, solids, granules or gelatin; (c) suspensions inan appropriate liquid; and (d) suitable emulsions. Tablet forms caninclude one or more of lactose, sucrose, mannitol, sorbitol, calciumphosphates, corn starch, potato starch, tragacanth, microcrystallinecellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellosesodium, talc, magnesium stearate, stearic acid, and other excipients,colorants, fillers, binders, diluents, buffering agents, moisteningagents, preservatives, flavoring agents, dyes, disintegrating agents,and pharmaceutically compatible carriers. Lozenge forms can comprise theactive ingredient in a flavor, usually sucrose and acacia or tragacanth,as well as pastilles comprising the active ingredient in an inert base,such as gelatin and glycerin or sucrose and acacia emulsions, gels, andthe like containing, in addition to the active ingredient, carriersknown in the art. It is recognized that the genetic vaccines, whenadministered orally, must be protected from digestion. This is typicallyaccomplished either by complexing the vaccine vector with a compositionto render it resistant to acidic and enzymatic hydrolysis or bypackaging the vector in an appropriately resistant carrier such as aliposome. Means of protecting vectors from digestion are well known inthe art. The pharmaceutical compositions can be encapsulated, e.g., inliposomes, or in a formulation that provides for slow release of theactive ingredient.

The packaged nucleic acids, alone or in combination with other suitablecomponents, can be made into aerosol formulations (e.g., they can be“nebulized”) to be administered via inhalation. Aerosol formulations canbe placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Suitable formulations for rectal administration include, for example,suppositories, which consist of the packaged nucleic acid with asuppository base. Suitable suppository bases include natural orsynthetic triglycerides or paraffin hydrocarbons. In addition, it isalso possible to use gelatin rectal capsules which consist of acombination of the packaged nucleic acid with a base, including, forexample, liquid triglycerides, polyethylene glycols, and paraffinhydrocarbons.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this invention, compositions can be administered, forexample, by intravenous infusion, orally, topically, intraperitoneally,intravesically or intrathecally. Parenteral administration andintravenous administration are the preferred methods of administration.The formulations of packaged nucleic acid can be presented in unit-doseor multi-dose sealed containers, such as ampoules and vials.

Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described. Cellstransduced by the packaged nucleic acid can also be administeredintravenously or parenterally.

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the patient over time. The dose will be determined by theefficacy of the particular vector employed and the condition of thepatient, as well as the body weight or vascular surface area of thepatient to be treated. The size of the dose also will be determined bythe existence, nature, and extent of any adverse side-effects thataccompany the administration of a particular vector, or transduced celltype in a particular patient.

In determining the effective amount of the vector to be administered inthe treatment or prophylaxis of an infection or other condition, thephysician evaluates vector toxicities, progression of the disease, andthe production of anti-vector antibodies, if any. In general, the doseequivalent of a naked nucleic acid from a vector is from about 1 μg to 1mg for a typical 70 kilogram patient, and doses of vectors used todeliver the nucleic acid are calculated to yield an equivalent amount oftherapeutic nucleic acid. Administration can be accomplished via singleor divided doses.

In therapeutic applications, compositions are administered to a patientsuffering from a disease (e.g., an infectious disease or autoimmunedisorder) in an amount sufficient to cure or at least partially arrestthe disease and its complications. An amount adequate to accomplish thisis defined as a “therapeutically effective dose.” Amounts effective forthis use will depend upon the severity of the disease and the generalstate of the patient's health. Single or multiple administrations of thecompositions may be administered depending on the dosage and frequencyas required and tolerated by the patient. In any event, the compositionshould provide a sufficient quantity of the proteins of this inventionto effectively treat the patient.

In prophylactic applications, compositions are administered to a humanor other mammal to induce an immune response that can help protectagainst the establishment of an infectious disease or other condition.

The toxicity and therapeutic efficacy of the genetic vaccine vectorsprovided by the invention are determined using standard pharmaceuticalprocedures in cell cultures or experimental animals. One can determinethe LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (thedose therapeutically effective in 50% of the population) usingprocedures presented herein and those otherwise known to those of skillin the art.

A typical pharmaceutical composition for intravenous administrationwould be about 0.1 to 10 mg per patient per day. Dosages from 0.1 up toabout 100 mg per patient per day may be used, particularly when the drugis administered to a secluded site and not into the blood stream, suchas into a body cavity or into a lumen of an organ. Substantially higherdosages are possible in topical administration. Actual methods forpreparing parenterally administrable compositions will be known orapparent to those skilled in the art and are described in more detail insuch publications as Remington's Pharmaceutical Science, 15th ed., MackPublishing Company, Easton, Pa. (1980).

The multivalent antigenic polypeptides of the invention, and geneticvaccines that express the polypeptides, can be packaged in packs,dispenser devices, and kits for administering genetic vaccines to amammal. For example, packs or dispenser devices that contain one or moreunit dosage forms are provided. Typically, instructions foradministration of the compounds will be provided with the packaging,along with a suitable indication on the label that the compound issuitable for treatment of an indicated condition. For example, the labelmay state that the active compound within the packaging is useful fortreating a particular infectious disease, autoimmune disorder, tumor, orfor preventing or treating other diseases or conditions that aremediated by, or potentially susceptible to, a mammalian immune response.

EXAMPLES

The following examples are offered to illustrate, but not to limit thepresent invention.

Example 1 Development of Broad-Spectrum Vaccines Against BacterialPathogens and Toxins

A. Evolution of Yersinia V-Antigens

This Example describes the use of DNA shuffling to develop immunogensthat produce strong cross-protective immune responses against a varietyof Yersinia strains. Passive immunization with anti-V-antigen antibodiesor active immunization with purified V-antigen can provide protectionfrom challenge with a virulent autologous Yersinia species. However,protection against heterologous species is limited (Motin et al. (1994)Infect. Immun. 62: 4192).

V-antigen genes from a variety of Yersinia strains, including serotypesof Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis are subjectedto DNA shuffling as described herein. The Yersinia pestis V antigencoding sequence, for example, is used as a query in a database search toidentify homologous genes that can be used in a family shuffling formatto obtain improved antigens. Results for a BLAST search of GenBank andEMBL databases are shown in Table 1, in which each line represents aunique sequence entry listing the database, accession number, locusname, bit score and E value. See, Altschul et al. (1997) Nucleic AcidsRes. 25:3389-3402, for a description of the search algorithm).Homologous antigens have been cloned and sequenced from a number ofrelated yet distinct Yersinia strains and additional natural diversityis obtained by cloning antigen genes from other strains. These genes andothers or fragments thereof are cloned by methods such as PCR, shuffledand screened for improved antigens. TABLE 1 Sequences producingsignificant alignments Score E Database/Accession No. Gene (bits) Valuegb|M26405|YEPLCR Yersinia pestis lcrG, lcrV, and lcrH genes, 1945 0.0 cogb|AF053946|AF053946 Yersinia pestis plasmid pCD1, complete pla 1945 0.0emb|X96802|YPTPIVANT Y. pseudotuberculosis V antigen gene 1834 0.0gb|M57893|YEPLCRGVHP Yersinia pseudotuberculosis V-antigen 1818 0.0gb|AF080155|AF080155 Yersinia enterocolitica pYV LcrV (lcrV) 1723 0.0antigen emb|X96801|YE96PVANT Y. enterocolitica V antigen gene, strain Y-. . . 1667 0.0 emb|X96799|YE108VANT Y. enterocolitica V antigen gene,strain Y- . . . 1659 0.0 emb|X96800|YE527VANT Y. enterocolitica Vantigen gene, serotype . . . 1651 0.0 emb|X96798|YE808VANT Y.enterocolitica V antigen gene, strain 1643 0.0 8081 emb|X96796|YE314VANTY. enterocolitica V antigen gene, strain WA. 1237 0.0emb|X96797|YENCTVANT Y. enterocolitica V antigen gene, strain 1221 0.0NCTC gb|S38727|S38727 lcrGVH operon: lcrV = V-antigen [Yersinia 3659e−99 pseudo.]

Shuffled clones are selected by phage display and/or screened by ELISAto identify those recombinant nucleic acids that encode polypeptidesthat have multiple epitopes corresponding to the different serotypes.The shuffled antigen genes are cloned into a filamentous phage genomefor polyvalent phage display or a suitable phagemid vector formonovalent phage display. A typical protocol for panning antigens byphage display is as follows.

-   -   Coat an appropriate surface (e.g., Nunc Maxisorp tube or        multiwell plate) at 4° C. overnight with the target antibody,        usually at a concentration of 1-10 μg/ml in PBS or other        suitable buffer    -   Rinse and Block with PBSM (PBS+3% nonfat dry milk) at 37° C. for        1-2 hr    -   Pre-block phage if needed (PBSM, RT 1 hr)    -   Rinse tube and allow phage to bind (usually 1 hr (37° C.)    -   Can vary time, temp, buffer, add a competitive inhibitor, etc.    -   Wash extensively (15×) with PBST (PBS+0.1% TWEEN20), then PBS    -   Elute bound phage with low pH (e.g., 10 mM glycine), 100 mM        triethylamine, competitive ligand, protease, etc. and then        neutralize pH if needed.    -   Infect E. coli with eluted phage to transduce expression        phagemid into new host. Titer and plate for colonies on drug        plates    -   Pool colonies into media, grow cells and infect with helper        phage to produce phage for next round

Phage ELISA assays are a useful method to rapidly evaluate single clonesafter panning of libraries. Single colonies are picked in individualwells of a multiwell plate containing 2YT media and grown as a masterplate. A replicate plate is infected with helper phage and grown so thatphage from a single well will display a single antigen variant. Asuitable protocol for phage ELISA assays is as follows.

-   -   Coat microtiter plate with 50 μl of 1 μg/ml target antibody        4° C. overnight    -   Rinse and block with PBSM for 2 hrs @ 37° C.    -   Rinse, add preblocked phage and allow to bind 1 hr @ 37° C.    -   Wash plates with PBST 3×, then PBS 3× with 2 min soaks    -   Add HRP(or AP)-conjugated anti-M13 antibodies for 1 hr @ 37° C.    -   Add substrate and measure absorbance    -   Identify positive clones for further evaluation

ELISA assays can also be used to screen for individual antigens withmultiple epitopes or increased expression levels. Single colonies arepicked in individual wells of a multiwell plate containing appropriatemedia and grown as a master plate so that antigens produced from asingle well are a single antigen variant. A replicate plate is grown andinduced for protein production, e.g., by addition of 0.5 mM IPTG for Lacrepressor-based systems and grown for an appropriate time for theantigen to be produced. At this point a crude antigen preparation ismade which depends on the antigen and where it is produced. Secretedproteins can be evaluated by assaying the cell supernatants aftercentrifugation. Periplasmic proteins are often readily released fromcells by simple extraction into hyper- or hypo-tonic buffers.Intracellularly produced proteins will require some form of cell lysissuch as detergent treatment to release them. A suitable protocol forELISA assays is as follows.

-   -   Coat microtiter plate with 50 μl of 1 μg/ml target antibody        4° C. overnight    -   Rinse and block with PBSM for 2 hrs @ 37° C.    -   Rinse, add antigen prep and allow to bind 1 hr @ 37° C.    -   Wash plates with PBST 3×    -   Add HRP(or AP)-conjugated secondary antibody and incubate for 1        hr @ 37° C.    -   Add appropriate substrate and measure absorbance    -   Identify positive clones for further evaluation

Antibodies specific for many of the various antigens are commerciallyavailable (e.g., Toxin Technology, Inc, Sarasota, Fla.) or can begenerated by immunizing suitable animals with purified antigens. ProteinA or Protein G Sepharose (Pharmacia) can be used to purifyimmunoglobulins from the serum. Various affinity purification schemescan be used to further purify family-specific antibodies if needed suchas immobilization of specific antigens to NHS-, CNBr-, orepoxy-activated sepharose beads. Other related antigens may be includedsoluble form to prevent binding and immobilization of cross-reactiveantibodies.

The multivalent polypeptides that are identified by the initialscreening protocol are purified and subjected to in vivo screening. Forexample, the shuffled antigens selected by a combination of any or noneof these methods are purified and used to immunize animals, initiallymice, which are then evaluated for improved immune responses. Typically10 micrograms of protein is injected to a suitable location with orwithout appropriate adjuvant, e.g., Alhydrogel (EM Seargent Pulp andChemical, Inc.) and the animals are boosted with an additional doseafter 2-4 weeks. At this point serum samples is drawn and evaluated byELISA assay for the presence of antibodies that cross-react againstmultiple parental antigens. In this ELISA assay format the antigens arecoated onto multiwell plates, then serial dilutions of each sera isallowed to bind. After washing unbound antibodies, a secondary HRP- orAP-conjugated antibody directed against the appropriate test antibodyconstant region, e.g., goat anti-mouse IgG Fc (Sigma) is bound. Afteranother washing, the appropriate substrate is added, e.g.,O-phenylenediamine (Sigma). The absorbance of each well is read by aplate reader at the appropriate wavelength (e.g., 490 nm for OPD) andthose producing high antibody titers to multiple antigens are selectedfor further evaluation.

Additionally, the ability of antigens to generate neutralizingantibodies can be evaluated in an appropriate system. Antigen variantsthat elicit a broad cross-reactive response are evaluated further in avirulent challenge model with the appropriate pathogenic organism. Forexample, the multivalent polypeptides are used to immunize mice, whichare then challenged with live Yersinia bacteria. Those multivalentpolypeptides that protect against the challenge are identified andpurified.

B. Evolution of Broad-Spectrum Vaccines Against Bacterial Toxins

This Example describes the use of DNA shuffling to obtain multivalentpolypeptides that are effective in inducing an immune response against abroad spectrum of bacterial toxins.

1. Staphylococcus

The Group A Streptococci, which can cause diseases such as foodpoisoning, toxic shock syndrome, and autoimmune disorders, are highlytoxic by inhalation. The family of Group A Streptococcus toxins numbersabout 30 related members, making this group a suitable target for familyshuffling. Accordingly, this Example describes the use of family DNAshuffling to create chimeric proteins that are capable of elicitingbroad spectrum protection.

Nucleic acids that encode many diverse attenuated toxins are subjectedto DNA shuffling as described herein. Table 2 shows the output of aBLAST search of GenBank, PDL, EMBL, and Swissprot using the S. aureusenterotoxin B protein to identify homologous genes that may be used in afamily shuffling format to obtain improved antigens. TABLE 2 Sequencesproducing significant alignments Score E Database/Accession No. Gene(bits) Value sp|P01552|ETXB_STAAU ENTEROTOXIN TYPE B PRECURSOR (SEB) >

554  e−157 pdb|1SE3| Staphylococcal Enterotoxin B Complex

504  e−142 Tri . . . pdb|1SEB|D Staphylococcus aureus >gi|1633348|pd

406  e−113 Staphyl . . . sp|P23313|ETC3_STAAU ENTEROTOXIN TYPE C-3PRECURSOR (SEC3) 376  e−103 sp|P01553|ETC1_STAAU ENTEROTOXIN TYPE C-1PRECURSOR (SEC1) 368  e−101 sp|P34071|ETC2_STAAU ENTEROTOXIN TYPE C-2PRECURSOR (SEC2) 361 2e−99 gi|295145 (L13376) enterotoxin[Staphylococcus 338 2e−92 gi|295151 (L13379) enterotoxin [Staphylococcus332 1e−90 gi|295143 (L13375) enterotoxin [Staphylococcus 330 4e−90gi|295149 (L13378) enterotoxin [Staphylococcus 329 1e−89 pdb|1JCK|BChain B, T-Cell Receptor Beta Chain

328 2e−89 With S . . . gi|295141 (L13374) enterotoxin [Staphylococcus328 3e−89 pdb|1SE2| Staphylococcal Enterotoxin C2, Monoc

327 4e−89 Ente . . . gi|1906052 (U91526) type C enterotoxin [Staphyl

326 8e−89 intermed . . . gi|295147 (L13377) enterotoxin [Staphylococcus323 7e−88 bbs|155101 enterotoxin=pyrogenic toxin [Staphyl

319 1e−86 4446, P . . . gi|476764 (L29565) superantigen [Streptococcus311 3e−84 gi|1245172 (U48792) superantigen SSA [Streptoco

310 4e−84 pyogenes] >. . . gi|1245174 (U48793) superantigen SSA[Streptoco

309 1e−83 pyogenes] sp|P08095|SPEA_STRPY EXOTOXIN TYPE A PRECURSOR(SCARLET F

225 2e−58 gi|47288 (X61560) type A exotoxin [Streptococ

211 3e−54 pyogenes] >gi|. . . pir||S18783 exotoxin type A precursor(allele 3) 211 4e−54 Streptococcu . . . pir|||S18786 exotoxin type Aprecursor (allele 2) 209 2e−53 Streptococcu . . . pir||S18789 exotoxin Aprecursor (allele 4) - Str 206 8e−53 pyo . . . gi|47328 (X61554) type Aexotoxin [Streptococc

196 9e−50 pyogenes] pir||A26152 streptococcal pyrogenic exotoxin type185 2e−46 precursor - . . . sp|P20723|ETXD_STAAU ENTEROTOXIN TYPE DPRECURSOR (SED) >

131 3e−30 sp|P13163|ETXA_STAAU ENTEROTOXIN TYPE A PRECURSOR (SEA) >

129 2e−29 prf||1704203A enterotoxin A [Staphylococcus aureus

128 3e−29 pdb|1ESF|A Staphylococcus aureus >gi|1633233|pd

125 2e−28 Staphyl . . . pir||A29566 enterotoxin A - Staphylococcus aureu

125 2e−28 sp|P12993|ETXE_STAAU ENTEROTOXIN TYPE E PRECURSOR (SEE) >

118 3e−26 gi|510692 (U11702) enterotoxin H [Staphylococc

98 7e−20 >gi|10 . . . gi|149047 (M94872) enterotoxin D [Plasmid pIB4

89 2e−17 gi|2689563 (U93688) enterotoxin [Staphylococcus 76 2e−13gi|153785 (M97156) pyrogenic exotoxin C [Strep

57 8e−08 pyogenes . . . sp|P13380|SPEC_STRPY EXOTOXIN TYPE C PRECURSOR(SPE C) 57 8e−08 gi|529754 (U02559) speC [Streptococcus pyogene

56 2e−07 pir||A30509 exotoxin C precursor - Streptococcus 56 2e−07 >gi|1. . . gi|529755 (U02560) speC [Streptococcus pyogene

55 4e−07 pir||S27240 enterotoxin B - Staphylococcus 53 1e−06 aureus(fragments)

Shuffled recombinant clones are initially selected by phage displayand/or screened by ELISA for the presence of multiple epitopes from thedifferent families. Variant proteins with multiple epitopes are purifiedand used to for in vivo screening as described above. The mouse sera areanalyzed for antibodies specific for different toxin subtypes andvariants that elicit broadly cross-reactive responses will be evaluatedfurther in challenge models.

2. Escherichia coli and Vibrio cholerae

This Example describes the use of DNA shuffling to obtain cross-reactivemultivalent polypeptides that induce an immune response against the E.coli heat-labile toxin (LT), cholera toxin (CT), and verotoxin (VT).Nucleic acids that encode cholera and LT toxin B-chains are subjected toDNA shuffling. Table 3 shows the results of a BLAST search using the V.cholerae toxin B-chain to identify homologous genes that can be used ina family shuffling format to obtain improved antigens. Homologousantigens have been cloned and sequenced from a number of related yetdistinct Vibrio and E. coli strains, and additional natural diversitycan be obtained by cloning antigen genes from other strains. These genesand others or fragments thereof can be cloned by methods such as PCR,shuffled and screened for improved antigens. TABLE 3 Sequences producingsignificant alignments Score E Database/Accession No. Gene (bits) Valuesp|P01556|CHTB_VIBCH CHOLERA ENTEROTOXIN, BETA CHAIN PREC

252 5e−67 gi|48890 (X58785) cholera toxin B protein (CT

248 8e−66 cholera . . . gi|758351 (X00171) ctx B [Vibrio cholerae] 2463e−65 prf|1001196A toxin, cholera [Vibrio cholerae] 246 3e−65gn1|PID|d1006853 (D30052) cholera toxin [Vibrio chole

244 1e−64 pir||XVVCB cholera enterotoxin chain B precurso

241 1e−63 cholerae gi|209556 (M23050) cholera toxin subunit B pre

228 7e−60 [Artificia . . . bbs|168005 holera-like enterotoxin B subunit[

211 1e−54 cholerae, . . . sp|P13811|ELBH_ECOLI HEAT-LABILE ENTEROTOXIN BCHAIN PREC

209 5e−54 pdb|1XTC|D Vibrio cholerae >gi|1827851|pdb|1XTC 207 2e−53choler . . . pdb|1FGB|D Vibrio cholerae >gi|1942839|pdb|1FGB 207 2e−53choler . . . pdb|2CHB|D Chain D, Cholera Toxin B-Pentamer Con

207 2e−53 With Gm1 . . . pdb|1CHP|D Vibrio cholerae >gi|1421512|pdb|1CHP205 1e−52 choler . . . pdb|1CHQ|D Vibrio cholerae >gi|14215126|pdb|1CHQ205 1e−52 choler . . . pdb|1CT1|D Chain D, Cholera Toxin B - PentamerMut 204 1e−52 Bound . . . sp|P32890|ELBP_ECOLI HEAT-LABILE ENLEROTOXIN BCHAIN PREC

204 2e−52 prt||0701264A toxin LTB Cistron, heat labile [Escher 201 9e−52coli] pir||QLECB heat-labile enterotoxin chain B prec

201 2e−51 Escheric . . . bbs|131495 (S60731) heat-labile enterotoxin B s

200 2e−51 B su . . . prf||770190A toxin [Vibrio cholerae] 199 6e−51pdb|1LTA|D Exchericnia coli >gi|494266|pdb|ILTA 179 4e−45 Escherichia C. . . pdb|1TET|P Vibrio cholerae 34 0.31

Those chimeric toxins that elicit high levels of neutralizing antibodiesagainst both toxins and have improved adjuvant properties areidentified. For example, shuffled clones are selected by phage displayand/or screened by ELISA assays for the presence of epitopes from thedifferent parental B-chains. Variants with multiple epitopes arepurified and further studied for their capacity to act as adjuvants andto elicit cross-protective immune responses in challenge models.

Example 2 Evolution of Broad-Spectrum Vaccines Against Borreliaburgdorferi

Lyme disease is currently one of the fastest-growing infectious diseasesin the United States. It is caused by infection of the spirochetebacterium Borrelia burgdorferi, which is carried and spread by the biteof infected ticks. Early signs of infection include skin rash andflu-like symptoms. If left untreated Lyme disease can cause arthritis,heart abnormalities, and facial paralysis. Treatment of early Lymedisease with antibiotics can stop the infection, but a lasting immunitymay not develop making reinfection possible. A current vaccine requiresthree immunizations over a 1-year period to acquire immunity.

Both passive and active immunization with the purified B. burgdorferiouter surface protein A (OspA) protein has been successful in protectingagainst infection with B. burgdorferi, but has no effect against ongoinginfections, since this antigen is not expressed in vertebrate hosts.OspA is normally anchored on the outside of the cell by a covalentlyattached lipid moiety through an amino terminal cysteine residue. Incontrast, the outer surface protein C (OspC) is highly expressed by thespirochete in vertebrate hosts and vaccination of infected individualswith OspC may be an effective therapeutic in curing the infection (Zhonget al. (1997) Proc. Nat'l. Acad. Sci. USA 94 12533-12538.

A recent BLAST search (Altschul, et al., (1997) Nucleic Acids Res.25:3389-3402) of the non-redundant GenBank, PDB, SwissProt, Spupdate,and PIR databases was used to identify homologues of the OspA outersurface protein gene. This resulted in the identification of over 200entries related to OspA. One hundred entries are shown in Table 4 belowfrom different strains of B. burgdorferi, B. garinii, B. afzelii, B.tanulkii, and B. turdi that share at least 83% DNA sequence identity tothe Borrelia burgdorferi OspA protein. The ospA genes from these andother strains provide a source of diversity for family shuffling toobtain improved antigens for the prevention of Lyme disease. These genesare cloned by methods such as PCR, shuffled and screened for improvedantigens. TABLE 4 Sequences producing significant alignments Score EDatabase/Accession No. Gene (bits) Value dbj|AB016977|AB016977 Borreliasp. gene for outer surface 1629 0.0 prote . . . dbj|AB016978|AB016978Borrelia sp. 10MT gene for outer 1614 0.0 surface . . .dbj|AB016975|AB016975 Borrelia turdi gene for outer surface 1526 0.0 pro. . . dbj|AB016976|AB016976 Borrelia sp. gene for outer surface 1187 0.0prote . . . gb|S48323|S48323 ospA = outer surface protein A (Borrelia948 0.0 burgdor . . . gb|L38657|BORFRA Borrelia burgdorferi (clone N3)ospA 948 0.0 gene frag . . . emb|X80186|BBPTROOPS B. burgdorferi PTroospA gene 938 0.0 emb|X65598|BBOSPA1 B. burgdorferi Osp A gene (TRO) 9380.0 gb|U20357|BBU20357 Borrelia burgdorferi C-1-11 outer 876 0.0 surfacepr . . . gb|S88693|S88693 outer surface protein A (Borrelia 858 0.0burgdorferi, . . . emb|X66065|BBOSPROA B. burgdorferi OspA gene forouter 839 0.0 surface p . . . emb|X85440|BGTISOSPA B. garinii ospA gene(TIsI substrain) 839 0.0 emb|X85438|BAPLJOSPA B. afzelii ospA gene (PLj7substrain) 837 0.0 <gi|9 . . . emb|X80183|BBPBOOSPA B. burgdorferi PBoospA gene 829 0.0 emb|Z29087|BBOSPAY B. burgdorferi (VS461) OspA genefor 821 0.0 outer su . . . emb|X85982|BADNAOSPA B. afzelii ospA gene 8210.0 emb|X62161|BBOSPAG B. burgdorferi plasmid ospA gene for 821 0.0outer su . . . gb|U78301|BBU78301 Borrelia afzelii major outer membrane821 0.0 surfac . . . emb|X65599|BBOSPA2 B. burgdorferi Osp A gene (PKO)819 0.0 emb|X70365|BBOPSAA B. burgdorferi OspA gene 819 0.0emb|X85439|BAPLUOSPA B. afzelii ospA gene (PLud substrain) 813 0.0emb|X81047|BBOPSA B. burgdorferi plasmid OspA gene 813 0.0gb|U20356|BAU20356 Borrelia afzelii BV1 outer surface 813 0.0 protein A. . . emb|X85437|BAPHOOSPA B. afzelii ospA gene (PHo substrain) 797 0.0emb|X80253|BBPWUDLL B. burgdorferi PWudll ospA gene 791 0.0emb|Z29086|BBOSPAX B. burgdorferi (G25) OspA gene for outer 791 0.0 surf. . . gb|L19702|BORMAJOSPR Borrelia burgdorferi outer surface 791 0.0protein . . . emb|X62387|BBSPA B. burgdorferi ospA gene for outer 7890.0 surface prot . . . emb|X60300|BBASPA B. burgdorferi gene for OspAouter 785 0.0 surface pro . . . emb|X62624|BBK48OSPA B. burgdorferi ospAgene 767 0.0 gb|M88764|BOROSPABA Borrelia burgdorferi operon major outer759 0.0 mem . . . emb|X63412|BBPOSPA B. burgdorferi plasmid ospA genefor 759 0.0 outer su . . . gb|L36036|BOROSPAL Borrelia burgdorferi outersurface 743 0.0 protein A . . . gb|U20358|BGU20358 Borrelia garinii LV4outer surface 743 0.0 protein A . . . emb|X85442|BB297OSPA B.burgdorferi ospA gene (297 substrain) 714 0.0 gb|L19701|BOROPSABBorrelia burgdorferi major outer 714 0.0 surface pro . . .emb|X14407|BBOSPAB Borrelia burgdorferi ospA and ospB 706 0.0 genes for. . . gb|AE000790|AE000790 Borrelia burgdorferi plasmid lp54, 706 0.0complet . . . gb|U20360|BBU20360 Borrelia burgdorferi S-1-10 outer 7060.0 surface pr . . . emb|X69606|BBKA0SPA B. burgdorferi 0spA gene 7060.0 >gi|1819262|gb|I284 . . . dbj|AB007100|AB007100 Borrelia gariniigene for outer surface 702 0.0 p . . . gb|M57248|BOROSPA B. burgdorfereiouter surface protein A 698 0.0 (OspA) . . . emb|X80182|BBPKAOPSA B.burgdorferi PKa ospA gene 698 0.0 dbj|AB007101|AB007101 Borrelia gariniigene for outer surface 694 0.0 p . . . emb|X85443|BBT25OSPA B.burgdorferi ospA gene (T255 692 0.0 substrain) emb|X16467|BBOSPABorrelia burgdorferi OspA gene for 690 0.0 outer surf . . .gb|U20359|BBU20359 Borrelia sp. LV5 outer surface protein 690 0.0 A pre. . . gb|AF026059|AF026059 Borrelia burgdorferi 50 kDa plasmid 682 0.0lipopr . . . emb|X85739|BBDNAOSPA B. burgdorferi ospA gene 682 0.0emb|X85441|BGWABOSPA B. garinii ospA gene (WABSou substrain) 676 0.0gb|U93709|U93709 Borrelia garinii outer surface protein 674 0.0 A (ospA. . . dbj|AB007099|AB007099 Borrelia garinii gene for outer surface 6620.0 p . . . emb|X80251|BBPHEIOSP B. burgdorferi PHei ospA gene 660 0.0gb|U49190|BGU49190 Borrelia garinii major outer membrane 652 0.0 surfac. . . emb|X65600|BBOSPA3 B. burgdorferi Osp A gene (HE) 652 0.0dbj|D29660|D29660 Borrelia burgdorferi gene for 652 0.0 outersurface pr. . . dbj|AB016979|AB016979 Borrelia valaisiana gene for outer 648 0.0surfac . . . dbj|AB007109|AB007109 Borrelia garinii gene for outersurface 646 0.0 p . . . gb|U93707|U93707 Borrelia garinii outer surfaceprotein 644 0.0 A (ospA . . . dbj|AB007102|AB007102 Borrelia gariniigene for outer surface 642 0.0 p . . . dbj|AB001041|AB001041 Borreliagarinii DNA for outer surface 636  e−180 pr . . . dbj|AB007114|AB007114Borrelia garinii gene for outer surface 632  e−179 p . . .dbj|AB007105|AB007105 Borrelia garinii gene for outer surface 632  e−179p . . . gb|U93710|U93710 Borrelia garinii outer surface protein 628 e−178 A (ospA . . . dbj|AB007106|AB007106 Borrelia garinii gene forouter surface 624  e−177 p . . . dbj|AB007104|AB007104 Borrelia gariniigene for outer surface 624  e−177 p . . . gb|U93706|U93706 Borreliagarinii outer surface protein 620  e−176 A (ospA . . .emb|X80256|BGPBROSPA B. garnii PBr ospA gene 613  e−173dbj|AB007108|AB007108 Borrelia garinii gene for outer surface 607  e−171p . . . gb|L81129|BOROSPAY Borrelia burgdorferi (isolate 2-1498 605 e−171 297) ou . . . gb|U93711|U93711 Borrelia garinii outer surfaceprotein 605  e−171 A (ospA . . . dbj|AB007103|AB007103 Borrelia gariniigene for outer surface 587  e−165 p . . . gb|L81128|BOROSPAZ Borreliaburgdorferi (isolate 2-1498 581  e−164 Son 188 . . . gb|L23137|BOROSPACBorrelia burgdorferi (27985CT2) OspA 577  e−162 gene, 3 . . .gb|L23139|BOROSPAE Borrelia burgdorferi (42373NY3) OspA 577  e−162 gene,3 . . . gb|L23142|BOROSPAI Borrelia burgdorferi (CA3) OspA gene, 577 e−162 3′end . . . gb|L23136|BOROSPAA Borrelia burgdorferi (BI9CT1) OspA577  e−162 gene, 3′e . . . gb|U93705|U93705 Borrelia garinii outersurface protein 569  e−160 A (ospA . . . gb|L23140|BOROSPAF Borreliaburgdorferi (41552MA) OspA 569  e−160 gene, 3′. . .dbj|AB007110|AB007110 Borrelia garinii gene for outer surface 565  e−159p . . . gb|L23143|BOROSPAJ Borrelia burgdorferi (CA7) OspA gene, 561 e−158 3′end . . . dbj|AB007112|AB007112 Borrelia garinii gene for outersurface 557  e−156 p . . . emb|X80254|BGT25OSPA B. garnii T25 ospA gene557  e−156 gb|U93708|U93708 Borrelia garinii outer surface protein 553 e−155 A (ospA . . . dbj|AB007107|AB007107 Borrelia garinii gene forouter surface 549  e−154 p . . . gb|L23141|BOROSPAH Borrelia burgdorferi(21343WI) OspA 545  e−153 gene, 3′. . . dbj|AB007111|AB007111 Borreliagarinii gene for outer surface 541  e−152 p . . . dbj|AB007113|AB007113Borrelia garinii gene for outer surface 537  e−151 p . . .gb|L23144|BOROSPAK Borrelia burgdorferi (CAB) OspA gene, 529  e−1483′end . . . gb|U78549|BAU78549 Borrelia afzelii major outer membrane 525 e−147 surfac . . . dbj|AB009863|AB009863 Borrelia garinii gene forouter surface 519  e−145 p . . . emb|X68059|BBOSPAGE B. burgdorferi OspAgene for outer 498  e−139 surface p . . . dbj|AB009862|AB009862 Borreliagarinii gene for outer surface 466  e−129 p . . . emb|X95360|BBOSPPFRAB. burgdorferi ospA gene (strain PFra) 460  e−127 emb|X68541|BBPHEI B.burgdorferi (PHEI) plasmid OspA gene 446  e−123 for ou . . .emb|X68540|BBPWUDI B. burgdorferi (PWudI) plasmid OspA gene 414  e−114for . . . emb|X95358|BGOSPPLI B. garinii ospA gene (strain PLi) 414 e−114 dbj|AB009860|AB009860 Borrelia garinii gene for outer surface 393 e−107 p . . . dbj|AB009858|AB009858 Borrelia garinii gene for outersurface 365 8e−99 p . . . dbj|AB009861|AB009861 Borrelia garinii genefor outer surface 283 2e−74 p . . .

A BLAST search with the B. burgdorferi OspC protein gene revealed over200 related entries. Entries for one hundred sequences sharing at least82% DNA sequence identity are shown in Table 5 below that provide asource of diversity for family shuffling to obtain improved therapeuticsin the treatment of Lyme disease. These genes are cloned by methods suchas PCR, shuffled and screened for improved antigens. TABLE 5 Sequencesproducing significant alignments Score E Database/Accession No. Gene(bits) Value gb|U04282|BBU04282 Borrelia burgdorfer GMP synthetase 12610.0 (guaA) g . . . gb|L42898|BOR31OSPC Borrelia burgdorfer (strain25015) 1124 0.0 outer s . . . gb|U01894|BBU01894 Borrelia burgdorfer B31outer surface 622  e−176 prote . . . dbj|D49497|BOROSPCA Borreliaburgdorfer gene for outer 622  e−176 surface . . . gb|AE000792|AE000792Borrelia burgdorfer plasmid cp26, 622  e−176 complet . . .emb|X69596|BBB3IOSPC B. burgdorferi ospC gene for outer 615  e−174surface . . . gb|AF029860|AF029860 Borrelia burgdorfer OC1 outer surface523  e−146 pro . . . gb|U91798|BBU91798 Borrelia burgdorfer strain L5outer 519  e−145 surface . . . gb|L42887|BOR20OSPC Borrelia burgdorfer(strain Ip2) outer 517  e−145 sur . . . gb|L81131|BOROSPCY Borreliaburgdorfer; substrain sensu 509  e−142 strict . . . gb|U91792|BBU91792Borrelia burgdorfer strain HII outer 478  e−133 surfac . . .gb|U91797|BBU91797 Borrelia burgdorfer strain IP3 outer 462  e−128surfac . . . gb|U91801|BBU91801 Borrelia burgdorfer strain PIF outer 444 e−123 surfac . . . dbj|AB001377|AB001377 Borrelia japonica strain NO67DNA for 430  e−118 Out . . . dbj|AB001378|AB001378 Borrelia japonicastrain OvKK7 DNA for 430  e−118 Ou . . . emb|X84783|BBOSPCTXW B.burgdorferi ospC gene (strain TXGW) 418  e−115 dbj|AB000355|AB000355Borrelia tanukii DNA for Outer surface 418  e−115 pr . . .gb|U91799|BBU91799 Borrelia burgdorfer strain IP1 outer 418  e−115surfac . . . dbj|AB001376|AB001376 Borrelia japonica strain Fi3Io DNAfor 414  e−114 Ou . . . emb|X73624|BBOSPCC B. burgdorferi (DK26) OspCgene 404  e−111 emb|X62162|BBPCG B. burgdorferi gene for pC protein 398 e−109 emb|X69590|BBWUDOSPC B. burgdorferi OspC gene, 3′ end 398  e−109emb|X81521|BAOSPC1 B. afzelii (strain PBo) ospC gene 377  e−102dbj|AB000345|AB000345 Borrelia afzelii DNA for Outer surface 365 6e−99pr . . . dbj|AB009900|AB009900 Borrelia afzelii gene for outer surface361 9e−98 p . . . dbj|AB009899|AB009899 Borrelia afzelii gene for outersurface 357 1e−96 p . . . emb|X81523|BAOSPC2 B. afzelii (strain PLj7)ospC gene 339 3e−91 gb|AF029871|AF029871 Borrelia burgdorfer OC12 outersurface 337 1e−90 pr . . . dbj|AB009897|AB009897 Borrelia afzelii genefor outer surface 337 1e−90 p . . . dbj|AB000354|AB000354 Borreliatanukii DNA for Outer surface 337 1e−90 pr . . . gb|L25413|BOROSPCBorrelia burgdorfer membrane protein 335 5e−90 (ospC) . . .dbj|D49502|BOROSPCF Borrelia afzelii gene for outer surface 335 5e−90pro . . . emb|X83555|BBDNAOSPC B. burgdorferi (B. pacificus strain) 3332e−89 ospC gene gb|L42874|BOR10OSPC Borrelia burgdorfer (strain Orth)331 8e−89 outer su . . . dbj|D49503|BOROSPCG Borrelia afzelii gene forouter surface 331 8e−89 pro . . . dbj|AB009894|AB009894 Borrelia afzeliigene for outer surface 329 3e−88 p . . . gb|L42890|BOR23OSPC Borreliaburgdorfer (strain E61) outer 329 3e−88 sur . . . gb|L42892|BOR25OSPCBorrelia burgdorfer (strain acal) 329 3e−88 outer su . . .dbj|D49501|BOROSPCE Borrelia afzelii gene for outer surface 327 1e−87pro . . . gb|U04240|BBU04240 Borrelia burgdorfer GMP synthetase 3171e−84 (guaA) a . . . gb|U04280|BBU04280 Borrelia burgdorfer GMPsynthetase 317 1e−84 (guaA) g . . . dbj|AB009901|AB009901 Borreliaafzelii gene for outer surface 309 3e−82 p . . . dbj|AB009893|AB009893Borrelia afzelii gene for outer surface 309 3e−82 p . . .gb|L42883|BOR17OSPC Borrelia burgdorfer (strain JSB) outer 307 1e−81 sur. . . gb|U04281|BBU04281 Borrelia burgdorfer HB19 outer surface 3055e−81 prot . . . dbj|AB009896|AB009896 Borrelia afzelii gene for outersurface 305 5e−81 p . . . emb|X81522|BBOSPC1 B. burgdorferi (strainPBre) ospC gene 297 1e−78 dbj|AB000349|AB000349 Borrelia afzelii DNA forOuter surface 297 1e−78 pr . . . emb|X73625|BBOSPCD B. burgdorferi (DK7)OspC gene 297 1e−78 dbj|D49509|BOROSPCM Borrelia garinii gene for outersurface 295 4e−78 pro . . . dbj|AB000346|AB000346 Borrelia afzelii DNAfor Outer surface 293 2e−77 pr . . . gb|AF029870|AF029870 Borreliaburgdorfer OC11 outer surface 289 3e−76 pr . . . gb|L42895|BOR28OSPCBorrelia burgdorfer (strain 28354) 289 3e−76 outer s . . .emb|X81524|BBOSPC2 B. burgdorferi (strain T255) ospC gene 289 3e−76dbj|D88296|D88296 Borrelia afzelii 26 kb circular plasmid 289 3e−76 DNAfo . . . emb|X81526|BGOSPC2 B. garinii (strain WABSou) ospC gene 2854e−75 >gi|8720 . . . emb|X84772|BBOSPCD32 B. garinii ospC gene (strainDK32) 285 4e−75 dbj|AB009902|AB009902 Borrelia afzelii gene for outersurface 285 4e−75 p . . . dbj|D49505|BOROSPC1 Borrelia garinii gene forouter surface 281 7e−74 pro . . . gb|U01892|BBU01892 Borrelia burgdorfer2591 outer surface 281 7e−74 prot . . . emb|X83552|BADNAOSPC B. afzelii(PLud strain) ospC gene 280 3e−73 dbj|D49378|BOROSPC64 Borrelia garinii(strain HT64) ospC 278 1e−72 gene f . . . dbj|D49379|BOROSPCVS Borreliaafzelli (strain VS461) ospC 274 2e−71 gene . . . gb|AF029864|AF029864Borrelia burgdorfer OC5 outer surface 266 4e−69 pro . . .dbj|AB000350|AB000350 Borrelia afzelii DNA for Outer surface 266 4e−69pr . . . gb|U08284|BBU08284 Borrelia burgdorfer 297 outer surface 2642e−68 prote . . . dbj|AB000343|AB000343 Borrelia afzelii DNA for Outersurface 260 2e−67 pr . . . dbj|AB000353|AB000353 Borrelia tanukii DNAfor Outer surface 258 1e−66 pr . . . emb|X84779|BBOSPCMUL B. burgdorferiospC gene (strain MUL) 256 4e−66 emb|X84768|BBOSPCD15 B. afzelii ospCgene (strain DK15) 256 4e−66 dbj|D88292|D88292 Borrelia garinii 26 kbcircular plasmid 254 2e−65 DNA fo . . . dbj|D49507|BOROSPCK Borreliagarinii gene for outer surface 254 2e−65 pro . . . emb|X83556|BGOSPCN34B. garinii (N34 strain) ospC gene 250 2e−64 gb|AF029866|AF029866Borrelia burgdorfer OC7 outer surface 250 2e−64 pro . . .dbj|D88294|D88294 Borrelia garinii 26 kb circular plasmid 250 2e−64 DNAfo . . . gb|L42888|BOR2IOSPC Borrelia burgdorfer (strain H9) outer 2489e−64 surf . . . gb|AF029862|AF029862 Borrelia burgdorfer OC3 outersurface 246 4e−63 pro . . . dbj|AB009891|AB009891 Borrelia afzelii genefor outer surface 246 4e−63 p . . . dbj|AB009898|AB009898 Borreliaafzelii gene for outer surface 246 4e−63 p . . . gb|AF029861|AF029861Borrelia burgdorfer OC2 outer surface 246 4e−63 pro . . .emb|X69593|BBTNOSPC B. burgdorferi OspC gene, 3′ end 246 4e−63dbj|D49377|BOROSPC57 Borrelia garinii (strain HT57) ospC 244 1e−62 genef . . . dbj|D49500|BOROSPCD Borrelia garinii gene for outer surface 2441e−62 pro . . . gb|L42896|BOR29OSPC Borrelia burgdorfer (strain 27579)242 6e−62 outer s . . . dbj|D49376|BOROSPCTC Borrelia garinii (strainTCLSK) ospC 240 2e−61 gene . . . gb|L42873|BOR9OSPC Borrelia burgdorfer(strain STMON) 238 9e−61 outer su . . . dbj|D49381|BOROSPC37 Borreliagarinii (strain HT37) ospC 238 9e−61 gene f . . . dbj|D49498|BOROSPCBBorrelia garinii gene for outer surface 238 9e−61 pro . . .emb|X69592|BBT25OSPC B. burgdorferi OspC gene, 3′ end 232 6e−59emb|X69594|BBPBROSPC B. burgdorferi OspC gene, 3′ end 228 9e−58dbj|D49506|BOROSPCJ Borrelia garinii gene for outer surface 220 2e−55pro . . . emb|X69595|BBPBIOSPC B. burgdorferi ospC gene, for outer 2202e−55 surface . . . emb|X83554|BGOSPPTRO B. garinii (PTrob strain) opsCgene 220 2e−55 emb|X73626|BBOSPCE B. burgdorferi (DK6) OspC gene 2202e−55 gb|L42870|BOR6OSPC Borrelia burgdorfer (strain VSDA) 220 2e−55outer sur . . . gb|L42894|BOR27OSPC Borrelia burgdorfer (strain 28691)218 8e−55 outer s . . . dbj|D49504|BOROSPCH Borrelia garinii gene forouter surface 208 8e−52 pro . . . gb|L42868|BOR4OSPC Borrelia burgdorfer(strain ZS7) outer 206 3e−51 surf . . . dbj|AB000358|AB000358 BorreliaJaponcia DNA for Outer surface 204 1e−50 p . . . dbj|AB000351|AB000351Borrelia Japoncia DNA for Outer surface 204 1e−50 p . . .

Example 3 Evolution of Broad-Spectrum Vaccines Against Mycobacterium

Tuberculosis is an ancient bacterial disease caused by Mycobacteriumtuberculosis that continues to be an important public health problemworldwide and calls are being made for an improved effort in eradication(Morb. Mortal Wkly Rep (1998 Aug. 21; 47(RR-13): 1-6). It infects over50 million people and over 3 million people will die from tuberculosisthis year. The currently available vaccine, Bacille Calmette-Guerin(BCG) is found to be less effective in developing countries and anincreasing number of multidrug-resistant (MDR) strains are beingisolated.

The major immunodominant antigen of M. tuberculosis is the 30-35 kDa(a.k.a. antigen 85, alpha-antigen) which is normally a lipoglycoproteinon the cell surface. Other protective antigens include a 65-kDa heatshock protein, and a 36-kDa proline-rich antigen (Tascon et al. (1996)Nat. Med. 2: 888-92).

Table 6 shows the output of a BLAST search using the 30-35 kDa major M.Tuberculosis antigen (a.k.a. antigen 85, alpha-antigen) coding sequenceto identify homologous genes that may be used in a family shufflingformat to obtain improved antigens. Many homologous antigens have beencloned and sequenced from a large number of related yet distinctmycobacterial strains. These genes are cloned by methods such as PCR,shuffled and screened for improved antigens. TABLE 6 Sequences producingsignificant alignments Score E Database/Accession No. Gene (bits) Valuegb|U38939|MTU38939 Mycobacterium tuberculosis 30 kDa 1939 0.0 extracellu. . . emb|X62398|MT85B M. tuberculosis (strain Eraman) gene for 1939 0.085-B a . . . emb|Z97193|MTCY180 Mycobacterium tuberculosis H37Rv 19390.0 complete ge . . . emb|X62397|MB85B M. bovis (strain 1173P2) gene for85-B 1931 0.0 antigen gb|M21839|MSGBCGA M. bovis BCG gene encodingalpha- 1869 0.0 antigen, comp . . . emb|X53897|MKAANTIG Mycobacteriumkansasii gene for alpha 819 0.0 antigen dbj|D26187|MSGAA Mycobacteriumscrofuraceum DNA for 706 0.0 alpha-antig . . . dbj|D16546|MSGAAGMycobacterium intracellulare gene for 581  e−164 alpha-a . . .emb|X63437|MAALANT M. avium gene for alpha-antigen 569  e−160dbj|D14253|MSGATCC139 Mycobacterium intracellulare DNA for 533  e−149alph . . . gb|L01095|MSGB38COS M. leprae genomic DNA sequence, cosmid371  e−100 B38 . . . emb|X60934|ML85BA M. leprae gene for 85-B antigen363 4e−98 emb|Z11666|MLFBPAPR M. leprae fibronectin-binding protein 3472e−93 antige . . . gb|M27016|MSG32KDA Mycobacterium tuberculosis 32 kDa317 2e−84 antigen gene. emb|X53034|MB32PG Mycobacterium bovis gene for32 kDa 317 2e−84 protein dbj|D26486|MSG32KDAP Mycobacterium bovis genesfor 32 kDa 317 2e−84 protei . . . emb|AL022076|MTV026 Mycobacteriumtuberculosis H37Rv 317 2e−84 complete g . . . gb|U47335|MTU47335Mycobacterium tuberculosis 317 2e−84 extracellular 32 . . .dbj|D78142|MSGBCGA85B Mycobacterium bovis gene for alpha 309 5e−82antige . . . emb|Y10378|MG85AANT M. gordonae gene encoding 85-A antigen303 3e−80 dbj|D78144|D78144 Mycobacterium avium gene for MPT51, 2582e−66 antigen 8 . . . gb|M90648|MSG85AA Mycobacterium leprae 85-Aantigen gene, 236 6e−60 compl . . . dbj|D43841|MSGA85CA Mycobacteriumleprae DNA for antigen 85 222 8e−56 com . . . emb|X92567|MMARI147 M.marinum gene for 32 kDa protein 218 1e−54 (partial) emb|Z33658|MA32KPI6M. avium (ATCC 19075) gene for 32 kDa 192 7e−47 protein . . .dbj|D87323|D87323 Mycobacterium avium gene for antigen 186 5e−45 85C and. . . emb|Z33657|MA32KPI5 M. avium (ATCC 15769) gene for 32 kDa 1842e−44 protein . . . emb|Z33662|MI32KPI10 M. intracellulare (ATCC 13950)gene for 168 1e−39 32 k . . . emb|X92566|MASIA122 M. asiaticum gene for32 KDa protein 168 1e−39 (partial) emb|Y07715|MA32KPRO1 M. asiaticumgene segment of 32-kDa 168 1e−39 protein emb|Z50760|MA32K511 M. aviumcomplex gene for 32 kDa protein 168 1e−39 (pa . . . emb|Z50759|MA32K1112M. avium complex gene for 32 kDa protein 167 4e−39 (p . . .emb|Z50767|MA32K769 M. avium complex gene for 32 kDa protein 161 3e−37(pa . . . emb|Z50774|MA32K966 M. avium complex gene for 32 kDa protein161 3e−37 (pa . . . emb|Z33659|MA32KPI7 M. avium (ATCC 19074) gene for32 kDa 161 3e−37 protein . . . emb|Z33661|MI32KPI9 M. intracellulare(ATTC 35762) gene for 161 3e−37 32 kD . . . emb|Z50763|MA32K559 M. aviumcomplex gene for 32 kDa protein 161 3e−37 (pa . . . emb|Z50772|MA32K961M. avium complex gene for 32 kDa protein 157 4e−36 (pa . . .emb|Z50765|MA32K576 M. avium complex gene for 32 kDa protein 153 6e−35(pa . . . emb|Z50770|MA32K904 M. avium complex gene for 32 kDa protein153 6e−35 (pa . . . emb|Z33667|MM32KPI15 M. malmoense gene for 32 kDaprotein 153 6e−35 (partial) emb|Z50768|MA32K814 M. avium complex genefor 32 kDa protein 153 6e−35 (pa . . . emb|Z50764|MA32K575 M. aviumcomplex gene for 32 kDa protein 149 1e−33 (pa . . . emb|Z50762|MA32K558M. avium complex gene for 32 kDa protein 145 2e−32 (pa . . .emb|X57229|MT85CG Mycobacterium tuberculosis gene for 145 2e−32 antigen8 . . . emb|Z50761|MA32K554 M. avium complex gene for 32 kDa protein 1452e−32 (pa . . . emb|Z92770|MTCI5 Mycobacterium tuberculosis H37Rv 1452e−32 complete geno . . . emb|X92570|MSZUL8 M. szulgai gene for 32 kDaprotein 141 2e−31 (partial) emb|Z50758|MA32K1076 M. avium complex genefor 32 kDa protein 127 4e−27 (p . . . emb|Z50766|MA32K577 M. aviumcomplex gene for 32 kDa protein 123 6e−26 (pa . . . emb|Z33654|MB32KPI2M. bovis (BCG) gene for 32 kDa protein 119 9e−25 (parti . . .emb|X92573|MTRIV151 M. triviale gene for 32 kDa protein 109 9e−22(partial) emb|X92583|MCEL1236 M. celatum gene for 32 kDa protein 1008e−19 (partial) emb|Z21950|ML85APRA M. leprae of 85A protein gene 908e−16 >gi|287923|emb . . . gb|L78816|MSGB26CS Mycobacterium lepraecosmid B26 DNA 88 3e−15 sequence. emb|Z21951|ML85CPRA M. leprae of 85Cprotein gene 88 3e−15 gb|M90649|MSG85CA Mycobacterium leprae 85-Cantigen gene, 88 3e−15 compl . . . emb|X92582|MCELI235 M. celatum genefor 32 kDa protein 86 1e−14 (partial) emb|X92577|MPHLE89 M. phlei genefor 32 kDa protein 82 2e−13 (partial) emb|X92581|MBRA1077 M. branderigene for 32 kDa protein 82 2e−13 (partial) emb|Y07718|MF32KPRO4 M.flavescens gene segment of 32-kDa 82 2e−13 protein emb|X92575|MFORT131M. fortuitum gene for 32 kDa protein 80 8e−13 (partial)emb|Z33663|MA32KPI11 M. avium-intracellulare complex gene for 76 1e−1132 . . . emb|X92576|MPERE132 M. peregrinum gene for 32 kDa protein 745e−11 (partial) emb|Z50776|MAH04894 M. avium complex gene for 32 kDaprotein 68 3e−09 (pa . . . emb|X92571|MXENO201 M. xenopi gene for 32 kDaprotein 68 3e−09 (partial) emb|Y07717|MS32KPRO3 M. smegmatis genesegment of 32-kDa 66 1e−08 protein emb|Z50769|MA32K822 M. avium complexgene for 32 kDa protein 60 7e−07 (pa . . . emb|Z50775|MAH03994 M. aviumcomplex gene for 32 kDa protein 60 7e−07 (pa . . . emb|Y07719|MV32KPRO5M. vaccae gene segment of 32-kDa protein 54 4e−05 emb|X92580|MVAC91 M.vaccae gene for 32 kDa protein 54 4e−05 (partial) emb|X92574|MNONC45 M.nonchromogenicum gene for 32 kDa 48 0.003 protein ( . . .emb|X92569|MSIMI95 M. simiae gene for 32 kDa protein 48 0.003 (partial)emb|AJ002150|MTAJ2150 Mycobacterium tuberculosis H37Rv, MPT51 46 0.011gene emb|Z79700|MTCY10D7 Mycobacterium tuberculosis H37Rv 44 0.043complete g . . . gb|M58472|ATUCAT A. tumefaciens chloramphenicol 42 0.17acetyltransferas . . . emb|X92578|MSMEG90 M. smegmatis gene for 32 kDaprotein 40 0.66 (partial) emb|X92572|MTER260 M. terrae gene for 32 kDaprotein 40 0.66 (partial) emb|X92568|MSCRO149 M. scrofulaceum gene for32 kDa protein 40 0.66 (par . . . emb|Z33666|MG32KPI14 M. gordonae (ATCC14470) gene for 32 kDa 40 0.66 pro . . . gb|M17700|FLCNPCA InfluenzaC/California/78 nucleoprotein 38 2.6 RNA ( . . .

Example 4 Evolution of Broad-Spectrum Vaccines Against Helicobacterpylori

Chronic infection of the gastroduodenal mucosae by Helicobacter pyloribacteria is responsible for chronic active gastritis, peptic ulcers, andgastric cancers such as adenocarcinoma and low-grade B-cell lymphoma. Anincreasing occurrence of antibiotic-resistant strains is limiting thistherapy. The use of vaccines to both prevent and treat ongoinginfections is being actively pursued (Crabtree J E (1998) Gut 43: 7-8;Axon A T (1998) Gut 43 Suppl 1: S70-3; Dubois et al. (1998) Infect.Immun. 66: 4340-6; Tytgat G N (1998) Aliment. Pharmacol. Ther. 12 Suppl1: 123-8; Blaser M J (1998) BMJ 316: 1507-10; Marchetti et al. (1998)Vaccine 16: 33-7; Kleanthous et al. (1998) Br. Med. Bull. 54: 229-41;Wermeille et al. (1998) Pharm. World Sci. 20: 1-17.

Identification of appropriate Helicobacter antigens for use inpreventive and therapeutic vaccines can include two-dimensional gelelectrophoresis, sequence analysis, and serum profiling (McAtee et al.(1998) Clin. Diagn. Lab. Immunol. 5:537-42; McAtee et al. (1998)Helicobacter 3: 163-9). Antigenic differences between relatedHelicobacter species and strains can limit the use of vaccines forprevention and treatment of infections (Keenan et al. (1998) FEMSMicrobiol Lett. 161: 21-7).

In this Example, DNA family shuffling of related yet immunologicallydistinct antigens allows for the isolation of complex chimeric antigensthat can provide a broad cross-reactive protection against many relatedstrains and species of Helicobacter. Mouse models of persistentinfection by mouse-adapted H. pylori strains that have been used toevaluate therapeutic use of vaccines against infection are used toevaluate shuffled antigens (Crabtree J E (1998) Gut 43: 7-8; Axon A T(1998) Gut 43 Suppl 1:S70-3).

The vacuolating cytotoxin (VacA) and cytotoxin associated gene products(CagA) have been evaluated as a vaccine against H. pylori infection inanimal models which supports the application of this approach in humans.

Table 7 shows the results of a BLAST search using the H. pylori VacAgene to identify homologous genes that can be used in a family shufflingformat to obtain improved antigens. Homologous antigens have been clonedand sequenced from a number of related yet distinct H. pylori strainsand additional natural diversity can be obtained by cloning antigengenes from other strains. These genes and others or fragments thereofare cloned by methods such as PCR, shuffled and screened for improvedantigens. TABLE 7 Sequences producing significant alignments Score EDatabase/Accession No. Gene (bits) Value gb|U95971|HPU95971 Helicobacterpylori 95-54 (J128) 7874 0.0 inactive cy . . . gb|AE000598|HPAE000598Helicobacter pylori section 76 of 2468 0.0 134 of . . .gb|AF001358|HPAF001358 Helicobacter pylori vacuolating 2405 0.0 cytotoxi. . . emb|Z26883|HPCYTTOX H. pylori gene for cytotoxin. 2389 0.0gb|U05677|HPU05677 Helicobacter pylori 87-203 2389 0.0 vacuolating cytot. . . gb|U05676|HPU05676 Helicobacter pylori 60190 2355 0.0cysteinyl-tRNA syn . . . gb|U29401|HPU29401 Helicobacter pylorivacuolating 2345 0.0 cytotoxin ho . . . emb|AJ006969|HPY6969Helicobacter pylori vacA gene, 2103 0.0 strain Mz28 . . .gb|S72494|S72494 140 kda cytotoxin Helicobacter 2050 0.0 pylori, Genomi. . . gb|U07145|HPU07145 Helicobacter pylori NCTC 11638 2050 0.0cysteinyl tRN . . . emb|AJ006968|HPY6968 Helicobacter pylori vacA gene,2032 0.0 strain Mz26 . . . emb|AJ006970|HPY6970 Helicobacter pylori vacAgene, 1992 0.0 strain Mz29 . . . gb|AF077939|AF077939 Helicobacterpylori strain 166 1834 0.0 vacuolating . . . gb|AF077940|AF077940Helicobacter pylori strain 539 1814 0.0 vacuolating . . .gb|AF077941|AF077941 Helicobacter pylori strain 549 1778 0.0 vacuolating. . . gb|AF077938|AF077938 Helicobacter pylori strain 50 1746 0.0vacuolating . . . gb|U63255|HPU63255 Helicobacter pylori vacuolating 8350.0 cytotoxin ge . . . gb|U63270|HPU63270 Helicobacter pylorivacuolating 819 0.0 cytotoxin ge . . . gb|U63272|HPU63272 Helicobacterpylori vacuolating 819 0.0 cytotoxin ge . . . gb|U63283|HPU63283Helicobacter pylori vacuolating 819 0.0 cytotoxin ge . . .gb|U63284|HPU63284 Helicobacter pylori vacuolating 819 0.0 cytotoxin ge. . . gb|U63262|HPU63262 Helicobacter pylori vacuolating 803 0.0cytotoxin ge . . . gb|U63268|HPU63268 Helicobacter pylori vacuolating803 0.0 cytotoxin ge . . . gb|U63273|HPU63273 Helicobacter pylorivacuolating 803 0.0 cytotoxin ge . . . gb|U63282|HPU63282 Helicobacterpylori vacuolating 803 0.0 cytotoxin ge . . . gb|U63259|HPU63259Helicobacter pylori vacuolating 795 0.0 cytotoxin ge . . .gb|U63276|HPU63276 Helicobacter pylori vacuolating 779 0.0 cytotoxin ge. . . gb|U63287|HPU63287 Helicobacter pylori vacuolating 779 0.0cytotoxin ge . . . gb|U63263|HPU63263 Helicobacter pylori vacuolating771 0.0 cytotoxin ge . . . gb|U63269|HPU63269 Helicobacter pylorivacuolating 771 0.0 cytotoxin ge . . . gb|U63286|HPU63286 Helicobacterpylori vacuolating 771 0.0 cytotoxin ge . . . gb|U63275|HPU63275Helicobacter pylori vacuolating 763 0.0 cytotoxin ge . . .gb|U63279|HPU63279 Helicobacter pylori vacuolating 763 0.0 cytotoxin ge. . . gb|U63277|HPU63277 Helicobacter pylori vacuolating 755 0.0cytotoxin ge . . . gb|U63280|HPU63280 Helicobacter pylori vacuolating755 0.0 cytotoxin ge . . . gb|U63265|HPU63265 Helicobacter pylorivacuolating 747 0.0 cytotoxin ge . . . gb|U63267|HPU63267 Helicobacterpylori vacuolating 747 0.0 cytotoxin ge . . . gb|U63281|HPU63281Helicobacter pylori vacuolating 741 0.0 cytotoxin ge . . .gb|U63261|HPU63261 Helicobacter pylori vacuolating 739 0.0 cytotoxin ge. . . gb|U63274|HPU63274 Helicobacter pylori vacuolating 739 0.0cytotoxin ge . . . gb|U63285|HPU63285 Helicobacter pylori vacuolating737 0.0 cytotoxin ge . . . emb|AJ009430|HPAJ9430 Helicobacter pylorivacA gene 737 0.0 (partial), . . . emb|AJ009435|HPAJ9435 Helicobacterpylori vacA gene 730 0.0 (partial), . . . emb|AJ009439|HPAJ9439Helicobacter pylori vacA gene 730 0.0 (partial), . . .gb|U63271|HPU63271 Helicobacter pylori vacuolating 724 0.0 cytotoxin ge. . . emb|AJ009418|HPAJ9418 Helicobacter pylori vacA gene 722 0.0(partial), . . . emb|AJ009422|HPAJ9422 Helicobacter pylori vacA gene 7220.0 (partial), . . . gb|U63256|HPU63256 Helicobacter pylori vacuolating716 0.0 cytotoxin ge . . . gb|U63266|HPU63266 Helicobacter pylorivacuolating 716 0.0 cytotoxin ge . . . emb|AJ009420|HPAJ9420Helicobacter pylori vacA gene 714 0.0 (partial), . . .emb|AJ009424|HPAJ9424 Helicobacter pylori vacA gene 714 0.0 (partial), .. . emb|AJ009431|HPAJ9431 Helicobacter pylori vacA gene 714 0.0(partial), . . . gb|U63260|HPU63260 Helicobacter pylori vacuolating 7080.0 cytotoxin ge . . . gb|U63278|HPU63278 Helicobacter pylorivacuolating 708 0.0 cytotoxin ge . . . emb|AJ009419|HPAJ9419Helicobacter pylori vacA gene 706 0.0 (partial), . . .emb|AJ009428|HPAJ9428 Helicobacter pylori vacA gene 706 0.0 (partial), .. . emb|AJ009437|HPAJ9437 Helicobacter pylori vacA gene 706 0.0(partial), . . . emb|AJ009427|HPAJ9427 Helicobacter pylori vacA gene 7040.0 (partial), . . . gb|U63257|HPU63257 Helicobacter pylori vacuolating700 0.0 cytotoxin ge . . . emb|AJ009423|HPAJ9423 Helicobacter pylorivacA gene 698 0.0 (partial), . . . emb|AJ009432|HPAJ9432 Helicobacterpylori vacA gene 692 0.0 (partial), . . . emb|AJ009417|HPAJ9417Helicobacter pylori vacA gene 688 0.0 (partial), . . .emb|AJ009421|HPAJ9421 Helicobacter pylori vacA gene 688 0.0 (partial), .. . emb|AJ009426|HPAJ9426 Helicobacter pylori vacA gene 688 0.0(partial), . . . emb|AJ009438|HPAJ9438 Helicobacter pylori vacA gene 6880.0 (partial), . . . gb|U63264|HPU63264 Helicobacter pylori vacuolating676 0.0 cytotoxin ge . . . emb|AJ009433|HPAJ9433 Helicobacter pylorivacA gene 666 0.0 (partial), . . . emb|AJ009425|HPAJ9425 Helicobacterpylori vacA gene 658 0.0 (partial), . . . gb|U63258|HPU63258Helicobacter pylori vacuolating 652 0.0 cytotoxin ge . . .emb|AJ009442|HPAJ9442 Helicobacter pylori vacA gene 626  e−177(partial), . . . emb|AJ009444|HPAJ9444 Helicobacter pylori vacA gene 626 e−177 (partial), . . . gb|U80068|HPU80068 Helicobacter pylori strain213, 622  e−175 vacuolating . . . emb|AJ009434|HPAJ9434 Helicobacterpylori vacA gene 618  e−174 (partial), . . . emb|AJ009441|HPAJ9441Helicobacter pylori vacA gene 603  e−170 (partial), . . .gb|AF035616|HPVCP2 Helicobacter pylori strain R34A 599  e−168vacuolating . . . emb|AJ009447|HPAJ9447 Helicobacter pylori vacA gene587  e−165 (partial), . . . emb|AJ009440|HPAJ9440 Helicobacter pylorivacA gene 563  e−158 (partial), . . . emb|AJ009436|HPAJ9436 Helicobacterpylori vacA gene 555  e−155 (partial), . . . emb|AJ009443|HPAJ9443Helicobacter pylori vacA gene 555  e−155 (partial), . . .emb|AJ009446|HPAJ9446 Helicobacter pylori vacA gene 555  e−155(partial), . . . gb|U80067|HPU80067 Helicobacter pylori strain 184, 553 e−155 vacuolating . . . gb|AF042735|AF042735 Helicobacter pylori JK22553  e−155 vacuolating cytot . . . emb|AJ009445|HPAJ9445 Helicobacterpylori vacA gene 547  e−153 (partial), . . . gb|AF035609|AF035609Helicobacter pylori strain R10A 547  e−153 vacuolatin . . .gb|AF042734|AF042734 Helicobacter pylori JK1 vacuolating 537  e−150cytoto . . . gb|AF035612|AF035612 Helicobacter pylori strain R26A 3479e−93 vacuolatin . . . gb|AF035613|AF035613 Helicobacter pylori strainR40A 323 1e−85 vacuolatin . . . emb|AJ006967|HPY6967 Helicobacter pylorivacA gene 317 8e−84 strain Mz19 . . . gb|AF035615|HPVCP1 Helicobacterpylori strain R34A 315 3e−83 vacuolating . . . gb|AF035614|AF035614Helicobacter pylori strain R50A 307 8e−81 vacuolatin . . .gb|U91578|HPU91578 Helicobacter pylori strain F37 vacA 109 4e−21 gene,pa . . . gb|U91579|HPU91579 Helicobacter pylori strain F79 vacA 1094e−21 gene, pa . . . gb|U91575|HPU91575 Helicobacter pylori strain F84vacA 107 1e−20 gene, pa . . . emb|Y14742|HPVACA26 Helicobacter pyloripartial vacA gene, 101 9e−19 stra . . . gb|U91577|HPU91577 Helicobacterpylori strain F94 vacA gene, 100 3e−18 pa . . . gb|U91576|HPU91576Helicobacter pylori strain F71 vacA gene, 100 3e−18 pa . . .gb|AF035610|AF035610 Helicobacter pylori strain R13A 94 2e−16 vacuolatin. . . gb|U91580|HPU91580 Helicobacter pylori strain F80 vacA gene, 928e−16 pa . . . gb|AF035611|AF035611 Helicobacter pylori strain R59A 928e−16 vacuolatin . . . emb|Y14744|HPVACA49 Helicobacter pylori partialvacA gene, 88 1e−14 stra . . .

Table 8 shows the results of a BLAST search using the H. pylori CagAgene to identify homologous genes that can be used in a family shufflingformat to obtain improved antigens. Homologous antigens have been clonedand sequenced from a number of related yet distinct H. pylori strainsand additional natural diversity can be obtained by cloning antigengenes from other strains. These genes and others or fragments thereofare be cloned by methods such as PCR, shuffled and screened for improvedantigens. TABLE 8 Sequences producing significant alignments Score EDatabase/Accession No. Gene (bits) Value gb|AF083352|AF083352Helicobacter pylori cytotoxin associated p . . . 7041 0.0gb|AE000569|HPAE000569 Helicobacter pylori section 47 of 134 of . . .5501 0.0 gb|L11714|HECMAJANT Helicobacter pylori major antigen gene 49760.0 sequ . . . emb|X70039|HPCAI H. pylori cai gene for cytotoxicityassociated 4294 0.0 . . . gb|U60176|HPU60176 Helicobacter pylori cagpathogenicity 4294 0.0 island . . . dbj|AB003397|AB003397 Helicobacterpylori DNA for CagA, 4274 0.0 complet . . . gb|U80066|HPU80066Helicobacter pylori strain 213, cytotoxin- 349 2e−93 as . . .gb|U80065|HPU80065 Helicobacter pylori strain 184, cytotoxin- 343 1e−91as . . . gb|AF043488|AF043488 Helicobacter pylori JK252 cytotoxicity 1784e−42 ass . . . gb|AF043487|AF043487 Helicobacter pylori JK25cytotoxicity 170 1e−39 asso . . . gb|AF043489|AF043489 Helicobacterpylori JK269 cytotoxicity 163 2e−37 ass . . . emb|X70038|HPCAIDUP H.pylori DNA duplication sequence within 159 4e−36 th . . .gb|AF043490|AF043490 Helicobacter pylori JK22 cytotoxicity 153 2e−34asso . . .

Example 5 Development of Broad-Spectrum Vaccines Against Malaria

This Example describes the use of DNA shuffling to generate improvedvaccines against malaria infection. An excellent target for evolution byDNA shuffling is the Plasmodium falciparum merozoite surface protein,MSP 1 (Hui et al. (1996) Infect. Immun. 64: 1502-1509). MSP 1 isexpressed on the surface of merozoites as an integral membrane protein.It is cleaved by parasite proteases just before and concomitant withrupture and release from infected cells. The cleavage appears to beobligatory for full function in MSP 1 binding to RBC receptors. Thecleaved fragments remain attached to the membrane of the merozoite.Other membrane proteins on merozoites also participate in the attachmentand specific invasion events. MSP 1 is a proven candidate for inclusionin a vaccine against the asexual blood stage of malaria.

The genes encoding MSP 1 can be isolated from various isolates ofPlasmodium falciparum merozoites by PCR technology. Related naturallyexisting genes can be additionally used to increase the diversity of thestarting genes. A library of shuffled MSP1 genes is generated by DNAshuffling, and this library is screened for induction of efficientimmune responses.

The screening can be done by injecting individual variants into testanimals, such as mice or monkeys. Either purified recombinant proteins,or DNA vaccines or viral vectors encoding the relevant genes areinjected. Typically, a booster injection is given 2-3 weeks after thefirst injection. Thereafter, the sera of the test animals are collectedand these sera are analyzed for the presence of antibodies that reduceinvasion of merozoites into uninfected erythrocytes (RBC). RBC areinfected by the merozoite, immediately inside the RBC, the merozoitedifferentiates into a ring and this matures to a schizont that containsseveral nascent daughter merozoites, which then burst out of theinfected cell, destroying it, and go on to attach and invade anotherRBC. In vivo, the merozoite is likely only extracellular for seconds. Invitro, any blockade of this event can dramatically reduce the level ofreinfection. Antibodies against MSP 1 bind to the surface of merozoitesthat are released from schizont infected RBC when they rupture andthereby reduce the ability of these merozoites to attach and engagecognate RBC receptors on the uninfected RBC surface. Merozoiteattachment is reduced, merozoite entry into new RBC is reduced, and thenumbers of newly invaded cells detected at the early ring stage istherefore reduced if the culture is examined several hours after theblockade of invasion test. In some assay formats a surrogate ofmerozoite invasion inhibition is to note the appearance of agglutinatedmerozoites, although this is an indirect measure of antibodies thatcause reduced invasion.

The shuffled antigens that induce the most potent antibody responsesreducing invasion of merozoites into uninfected erythrocytes areselected for further testing and can be subjected to new rounds ofshuffling and selection. In subsequent studies, the capacity of theseantigens to induce antibodies in man is investigated. Again, eitherpurified recombinant antigens, or DNA vaccines or viral vectors encodingthe relevant genes are injected and the protective immune responses areanalyzed.

Example 6 Development of Broad-Spectrum Vaccines Against Viral Pathogens

This Example describes the use of DNA shuffling to obtain vaccines thatcan induce an immune response against multiple isolates of viralpathogens.

A. Venezuelan Equine Encephalitis Virus (VEE)

VEE belongs to the alphavirus genus, which are generally transmitted bymosquitoes. However, VEE is an unusual alphavirus in that it is alsohighly infectious by aerosol inhalation for both humans and rodents. Thedisease manifestations in humans range from subclinical or mild febriledisease to serious infection and inflammation of the central nervoussystem. Virus clearance coincides that of production of specificanti-VEE antibodies, which are believed to be the primary mediators ofprotective immune responses (Schmaljohn et al. (1982) Nature 297: 70).VEE is an unusual virus also because its primary target outside thecentral nervous system is the lymphoid tissue, and therefore,replication defective variants may provide means to target vaccines orpharmaceutically useful proteins to the immune system.

At least seven subtypes of VEE are known that can be identifiedgenetically and serologically. Based on epidemiological data, the virusisolates fall into two main categories: I-AB and I-C strains, which areassociated with VEE epizootics/epidemics, and the remaining serotypes,which are associated primarily with enzootic vertebrate-mosquito cyclesand circulate in specific ecological zones (Johnston and Peters, InFields Virology, Third Edition, eds. B. N. Fields et al.,Lippincott-Raven Publishers, Philadelphia, 1996).

The envelope protein (E) appears to be the major antigen in inducingneutralizing Abs. Accordingly, DNA shuffling is used to obtain a libraryof recombinant E proteins by shuffling the corresponding genes derivedfrom various strains of VEE. These libraries and individualschimeras/mutants thereof are subsequently screened for their capacity toinduce widely cross-reacting and protective Ab responses.

B. Flaviviruses

Japanese encephalitis virus (JE), Tick-borne encephalitis virus (TBE)and Dengue virus are arthropod-borne viruses belonging to the Flavivirusfamily, which comprises 69 related viruses. The heterogeneity of theviruses within the family is a major challenge for vaccine development.For example, there are four major serotypes of Dengue virus, and atetravalent vaccine that induces neutralizing Abs against all fourserotypes is necessary. Moreover, non-neutralizing antibodies induced byinfection or vaccination by one Dengue virus may cause enhancement ofthe disease during a subsequent infection by another serotype.Therefore, cross-protective, broad spectrum vaccines for TBE and JEwould provide significant improvements to the existing vaccines. In thisExample, the ability of DNA shuffling to efficiently generate chimericand mutated genes is used to generate cross-protective vaccines.

1. Japanese Encephalitis Virus

Japanese encephalitis virus (JE) is a prototype of the JE antigeniccomplex, which comprises St. Louis encephalitis virus, Murray Valleyencephalitis virus, Kunjin virus and West Nile virus (Monath and Heinz,In Fields Virology, Third Edition, eds. B. N. Fields et al.,Lippincott-Raven Publishers, Philadelphia, pp 961-1034, 1996).Infections caused by JE are relatively rare, but the case-fatality is5-40% because no specific treatment is available. JE is widelydistributed in China, Japan, Philippines, far-eastern Russia and Indiaproviding a significant threat to those traveling in these areas.Currently available JE vaccine is produced from brain tissues of miceinfected with single virus isolate. Side effects are observed in 10% to30% of the vaccines.

To obtain chimeric and/or mutated antigens that provide a protectiveimmune response against all or most of the viruses within the JEcomplex, DNA shuffling is performed on viral envelope genes. The aminoacid identity within the JE complex varies between 72% and 93%. Inaddition, significant antigenic variation has been observed among JEstrains by neutralization assays, agar gel diffusion, antibodyabsorption and monoclonal antibody analysis (Oda (1976) Kobe J. Med.Sci. 22: 123; Kobyashi et al. (1984) Infect. Immun. 44: 117). Moreover,the amino acid divergence of the envelope protein gene among 13 strainsfrom different Asian countries is as much as 4.2% (Ni and Barrett (1995)J. Gen. Virol. 76: 401). The resulting library of recombinantpolypeptides encoded by the shuffled genes is screened to identify thosethat provide a cross-protective immune response.

2. Tick-Borne Encephalitis Virus

The tick-borne encephalitis virus complex comprises 14 antigenicallyrelated viruses, eight of which cause human disease, including Powassan,Louping ill and Tick-borne encephalitis virus (TBE) (Monath and Heinz,In Fields Virology, Third Edition, eds. B. N. Fields et al.,Lippincott-Raven Publishers, Philadelphia, pp. 961-1034, 1996). TBE hasbeen recognized in all Central and Eastern European countries,Scandinavia and Russia, whereas Powassan occurs in Russia, Canada andthe United States. The symptoms vary from flu-like illness to severemeningitis, meningoencephalitis and meningoencephalitis with a fatalityrate of 1% to 2% (Gresikova and Calisher, In Monath ed., Thearboviruses: ecology and epidemiology, vol. IV, Boca Raton, Fla., CRCPress, pp. 177-203, 1988).

Family DNA shuffling is used to generate chimeric envelope proteinsderived from the TBE complex to generate crossprotective antigens. Theenvelope proteins within the family are 77-96% homologous, and virusescan be distinguished by specific mAbs (Holzmann et al., Vaccine, 10,345, 1992). The envelope protein of Powassan is 78% identical at theamino acid level with that of TBE, and cross-protection is unlikely,although epidemiological data is limited.

Langat virus is used as a model system to analyze protective immuneresponses in vivo (Iacono-Connors et al. (1996) Virus Res. 43: 125).Langat virus belongs to the TBE complex, and can be used in challengestudies in BSL3 facilities. Serological studies based on recombinantenvelope proteins are performed to identify immunogen variants thatinduce high levels of antibodies against envelope proteins derived frommost or all viruses of the TBE complex.

3. Dengue Viruses

Dengue viruses are transmitted though mosquito bites, posing asignificant threat to troops and civilian populations particularly intropical areas. There are four major serotypes of Dengue virus, namelyDengue 1, 2, 3 and 4. A tetravalent vaccine that induces neutralizingantibodies against all four strains of Dengue is required to avoidantibody-mediated enhancement of the disease when the individualencounters the virus of the other strain.

The envelope protein of Dengue virus has been shown to provide an immuneresponse that protects from a future challenge with the same strain ofvirus. However, the levels of neutralizing antibodies produced arerelatively low and protection from live virus challenge is not alwaysobserved. For example, mice injected with genetic vaccines encodingenvelope protein of Dengue-2 virus developed neutralizing antibodieswhen analyzed by in vitro neutralization assays, but the mice did notsurvive the challenge with live Dengue-2 virus (Kochel et al. (1997)Vaccine 15: 547-552). However, protective immune responses were observedin mice immunized with recombinant vaccinia virus expressing Dengue 4virus structural proteins (Bray et al. (1989) J. Virol. 63: 2853). Thesestudies indicate that vaccinations with E proteins work, but significantimprovements in the immunogenicity of the protective antigens arerequired.

In this Example, DNA shuffling is performed on the genes encoding theenvelope (E) protein from all four Dengue viruses and their antigenicvariants. Family DNA shuffling is used to generate chimeric E proteinvariants that induce high titer neutralizing antibodies against allserotypes of Dengue. The E proteins of the different dengue virusesshare 62% to 77% of their amino acids. Dengue 1 and Dengue 3 are mostclosely related (77% homologous), followed by Dengue 2 (69%) and Dengue4 (62%). These homologies are well in the range that allows efficientfamily shuffling (Crameri et al. (1998) Nature 391: 288-291).

The shuffled antigen sequences are incorporated into genetic vaccinevectors, the plasmids purified, and subsequently injected into mice. Thesera are collected from the mice and analyzed for the presence of highlevels of cross-reactive antibodies. The best antigens are selected forfurther studies using in vivo challenge models to screen forchimeras/mutants that induce cross-protection against all strains ofDengue.

C. Improved Expression and Immunogenicity of Hantaan Virus Glycoproteins

One of the advantages of genetic vaccines is that vectors expressingpathogen antigens can be generated even when the given pathogen cannotbe isolated in culture. An example of such potential situation was anoutbreak of severe respiratory disease among rural residents of theSouthwestern United States which was caused by a previously unknownhantavirus, Sin Nombre virus (Hjelle et al. (1994) J. Virol. 68: 592).Much RNA sequence information of the virus was obtained well before thevirus could be isolated and characterized in vitro. In these situations,genetic vaccines can provide means to generate efficient vaccines in ashort period of time by creating vectors encoding antigens encoded bythe pathogen. However, genetic vaccines can only work if these antigenscan be properly expressed in the host.

Hantaan virus belongs to the Bunyavirus family. A characteristic featureof this family is that their glycoproteins typically accumulate at themembranes of the Golgi apparatus when expressed by cloned cDNAs, therebyreducing the efficacy of corresponding genetic vaccines (Matsuoka et al.(1991) Curr. Top. Microb. Immunol. 169: 161-179). Poor expression ofHantaan virus glycoproteins on the cell surface is also one explanationfor poor immune responses following injections of Hantaan virus geneticvaccines.

In this Example, family DNA shuffling is used to generate recombinantHantaan virus derived glycoproteins that are efficiently expressed inhuman cells and that can induce protective immune responses against thewild-type pathogen. Nucleic acids that encode the Hantaan virusglycoprotein are shuffled with genes that encode other homologousBunyavirus glycoproteins. The resulting library is screened to identifyproteins that are readily expressed in human cells. The screening isperformed using a dual marker expression vector that enablessimultaneous analysis of transfection efficiency and expression offusion proteins that are PIG-linked to the cell surface (Whitehorn etal. (1995) Biotechnology (N Y) 13:1215-9).

Flow cytometry based cell sorting is used to select Hantaan virusglycoprotein variants that are efficiently expressed in mammalian cells.The corresponding sequences are then obtained by PCR or plasmidrecovery. These chimeras/mutants are further analyzed for their capacityto protect wild mice against Hantaan virus infections.

Example 7 DNA Shuffling of HSV-1 And HSV-2 Glycoproteins B and/or D asMeans to Induce Enhanced Protective Immune Responses

This Example describes the use of DNA shuffling to obtain HSVglycoprotein B (gB) and glycoprotein D (gD) polypeptides that exhibitimproved ability to induce protective immune responses uponadministration to a mammal. Epidemiological studies have shown thatprior infections with HSV-1 give partial protection against infectionswith HSV-2, indicating existence of cross-reactive immune responses.Based on previous vaccination studies, the main immunogenicglycoproteins in HSV appear to be gB and gD, which are encoded by 2.7 kband 1.2 kb genes, respectively. The gB and gD genes of HSV-1 are about85% identical to the corresponding gene of HSV-2, and the gB genes ofeach share little sequence identity with the gD genes. Baboon HSV-2 gBis appr. 75% identical to human HSV-1 or -2 gB, with rather longstretches of almost 90% identity. In addition, 60-75% identity is foundin portions of the genes of equine and bovine herpesviruses.

Family shuffling is employed using as substrates nucleic acids thatencode gB and/or gD from HSV-1 and HSV-2. Preferably, homologous genesare obtained from HSVs of various strains. An alignment of gD nucleotidesequences from HSV-1 and two strains of HSV-2 is shown in FIG. 7.Antigens encoded by the shuffled nucleic acids are expressed andanalyzed in vivo. For example, one can screen for improved induction ofneutralizing antibodies and/or CTL responses against HSV-1/HSV-2. Onecan also detect protective immunity by challenging mice or guinea pigswith the viruses. Screening can be done using pools or individualsclones.

Example 8 Evolution of HIV Gp120 Proteins for Induction of BroadSpectrum Neutralizing Ab Responses

This Example describes the use of DNA shuffling to generate immunogensthat crossreact among different strains of viruses, unlike the wild-typeimmunogens. Shuffling two kinds of envelope sequences can generateimmunogens that induce neutralizing antibodies against a third strain.

Antibody-mediated neutralization of HIV-1 is strictly type-specific.Although neutralizing activity broadens in infected individuals overtime, induction of such antibodies by vaccination has been shown to beextremely difficult. Antibody-mediated protection from HIV-1 infectionin vivo correlates with antibody-mediated neutralization of virus invitro.

FIG. 8 illustrates the generation of libraries of shuffled gp120 genes.gp120 genes derived from HIV-1DH12 and HIV-1IIIB(NL43) are shuffled. Thechimeric/mutant gp120 genes are then analyzed for their capacity toinduce antibodies that have broad spectrum capacity to neutralizedifferent strains of HIV. Individual shuffled gp120 genes areincorporated into genetic vaccine vectors, which are then introduced tomice by injection or topical application onto the skin. These antigenscan also be delivered as purified recombinant proteins. The immuneresponses are measured by analyzing the capacity of the mouse sera toneutralize HIV growth in vitro. Neutralization assays are performedagainst HIV-1DH12, HIV-1IIIB and HIV-189.6. The chimeras/mutants thatdemonstrate broad spectrum neutralization are chosen for further roundsof shuffling and selection. Additional studies are performed in monkeysto illustrate the capacity of the shuffled gp120 genes to provideprotection for subsequent infection with immunodeficiency virus.

Example 9 Antigen Shuffling of the Hepadnavirus Envelope Protein

The Hepatitis B virus (HBV) is one of a member of a family of virusescalled hepadnaviruses. This Example describes the use of genomes andindividual genes from this family are used for DNA shuffling, whichresults in antigens having improved properties.

A. Shuffling of Hepadnavirus Envelope Protein Genes

The envelope protein of the HBV assembles to form particles that carrythe antigenic structures collectively known as the Hepatitis B surfaceantigen (HBsAg; this term is also used to designate the protein itself).Antibodies to the major antigenic site, designated the “a” epitope(which is found in the envelope domain called S), are capable ofneutralizing the virus. Immunization with the HBsAg-bearing protein thusserves as a vaccine against viral infection. The HBV envelope alsocontains other antigenic sites that can protect against viral infectionand are potentially vital components of an improved vaccine. Theepitopes are part of the envelope protein domains known as preS1 andpreS2 (FIG. 9).

DNA shuffling of the envelope gene from several members of thehepadnavirus family is used to obtain more immunogenic proteins.Specifically, the genes from the following hepatitis viruses areshuffled:

-   -   a the human HBV viruses, subtypes ayw and adw2    -   a hepatitis virus isolated from chimpanzee    -   a hepatitis virus isolated from gibbon    -   a hepatitis virus isolated from woodchuck

If desired, genes from other genotypes of the human virus are availablefor inclusion in the DNA shuffling reactions. Likewise, other animalhepadnaviruses are available.

To promote the efficiency of the formation of chimeras resulting fromDNA shuffling, some artificial genes are made:

-   -   In one case, a synthetic gene is made that contains the HBV        envelope sequences, except for those codons which specify amino        acids found in the chimpanzee and gibbon genes. For those        codons, the chimpanzee or gibbon sequence is used.    -   In a second case, a synthetic gene is synthesized in which the        preS2 gene sequence from the human HBV adw2 strain is fused with        the woodchuck S region.    -   In a third case, all the oligonucleotides required to chemically        synthesize each of the hepadnavirus envelope genes are mixed in        approximately equal quantities and allowed to anneal to form a        library of sequences.

After DNA shuffling of the hepadnavirus envelope genes, either or bothof two strategies are used to obtain improved HBsAg antigens.

Strategy A: Antigens are screened by immunizing mice using two possiblemethods. The genes are injected in the form of DNA vaccines, i.e.,shuffled envelope genes carried by a plasmid that comprises the geneticregulatory elements required for expression of the envelope proteins.Alternatively, the protein is prepared from the shuffled genes and usedas the immunogen.

The sequences that give rise to greater immunogenicity for either thepreS1-, preS2- or S-borne HBV antigens are selected for a second roundof shuffling (FIG. 10). For the second round, the best candidates arechosen based on their improved antigenicity and their other propertiessuch as higher expression level or more efficient secretion. Screeningand further rounds of shuffling are continued until a maximumoptimization for one of the antigenic regions is obtained.

The individually optimized genes are then used as a combination vaccinefor the induction of optimal responses to preS1-, preS2- and S-bornepitopes.

Strategy B: After isolation of the individually optimized genes as inStrategy A, the preS1, preS2 and S candidates are shuffled together, orin a pairwise fashion, in further rounds to obtain genes which encodeproteins that demonstrate improved immunogenicity for at least tworegions containing HBsAg epitopes (FIG. 11).

B. Use of HBsAg to Carry Epitopes from Unrelated Antigens

Several of the characteristics of the HBsAg make it a useful protein tocarry epitopes drawn from other, unrelated antigens. The epitopes can beeither B epitopes (which induce antibodies) or T epitopes drawn from theclass I type (which stimulate CD8⁺ T lymphocytes and induced cytotoxiccells) or class II type (which induce helper T lymphocytes and areimportant in providing immunological memory responses.

1. B Cell Epitopes

Amino acid sequences of potential B epitopes are chosen from anypathogen. Such sequences are often known to induce antibodies, but theimmunogenicity is weak or otherwise unsatisfactory for preparation of avaccine. These sequences can also be mimotopes, which have been selectedbased on their ability to have a certain antigenicity or immunogenicity.

The amino acid coding sequences are added to a hepadnavirus envelopegene. The heterologous sequences can either replace certain envelopesequences, or be added in addition to all the envelope sequences. Theheterologous epitope sequences can be placed at any position in theenvelope gene. A preferred position is the region of the envelope genethat encodes the major “a” epitope of the HBsAg (FIG. 12). This regionis likely to be exposed on the external side of the particles formed bythe envelope protein, and thus will expose the heterologous epitopes.

DNA shuffling is carried out on the envelope gene sequences, keeping thesequence of the heterologous epitopes constant. Screening is carried outto choose candidates that are secreted into the culture medium aftertransfection of plasmids from the shuffled library into cells in tissueculture.

Clones that encode a secreted protein are then tested for immunogenicityin mice either as a DNA vaccine or as a protein antigen, as describedabove. Clones that give an improved induction of antibodies to theheterologous epitopes are chosen for further rounds of DNA shuffling.The process is continued until the immunogenicity of the heterologousepitope is sufficient for use as a vaccine against the pathogen fromwhich the heterologous epitopes were derived.

2. Class I Epitopes

MHC Class I epitopes are relatively short, linear peptide sequences thatare generally between 6 and 12 amino acids amino acids in length, mostoften 9 amino acids in length. These epitopes are processed byantigen-presenting cells either after synthesis of the epitope withinthe cell (usually as part of a larger protein) or after uptake ofsoluble protein by the cells.

Polynucleotide sequences that encode one or more class I epitopes areinserted into the sequence of a hepadnavirus envelope gene either byreplacing certain envelope sequences, or by inserting the epitopesequences into the envelope gene. This is typically done by modifyingthe gene before DNA shuffling or by including in the shuffling reactioncertain oligonucleotide fragments that encode the heterologous epitopesas well as sufficient flanking hepadnavirus sequences to be incorporatedinto the shuffled products.

Preferably, the heterologous class I epitopes are placed into differentpositions in the several hepadnavirus genes used for the DNA shufflingreaction. This will optimize the chances for finding chimeric genecarrying the epitopes in an optimal position for efficient presentation.

3. Class II Epitopes

MHC Class II epitopes are generally required to be part of a proteinwhich is taken up by antigen presenting cells, rather than synthesizedwithin the cell. Preferably, such epitopes are incorporated into acarrier protein such as the HBV envelope that can be produced in asoluble form or which can be secreted if the gene is delivered in theform of a DNA vaccine.

Polynucleotides that encode heterologous class II epitopes are insertedinto regions of the hepadnavirus envelope genes that are not involved inthe transmembrane structure of the protein. DNA shuffling is performedto obtain a secreted protein that also carries the class II epitopes.When injected as a protein, or when the gene is delivered as a DNAvaccine, the protein can be taken up by antigen presenting cells forprocessing of the class II epitopes.

Example 10 Evolution of Broad Spectrum Vaccines Against Hepatitis CVirus

Antigenic heterogeneity of different strains of Hepatitis C Virus (HCV)is a major problem in development of efficient vaccines against HCV.Antibodies or CTLs specific for one strain of HCV typically do notprotects against other strains. Multivalent vaccine antigens thatsimultaneously protect against several strains of HCV would be of majorimportance when developing efficient vaccines against HCV.

The HCV envelope genes, which encode envelope proteins E1 and E2, havebeen shown to induce both antibody and lymphoproliferative responsesagainst these antigens (Lee et al. (1998) J. Virol. 72: 8430-6), andthese responses can be optimized by DNA shuffling. The hypervariableregion 1 (HVR1) of the envelope protein E2 of HCV is the most variableantigenic fragment in the whole viral genome and is primarilyresponsible for the large inter- and intra-individual heterogeneity ofthe infecting virus (Puntoriero et al. (1998) EMBO J. 17: 3521-33).Therefore, the gene encoding E2 is a particularly useful target forevolution by DNA shuffling.

DNA shuffling of HCV antigens, such as nucleocapsid or envelope proteinsE1, E2, provides a means to generate multivalent HCV vaccines thatsimultaneously protect against several strains of HCV. These antigensare shuffled using the family DNA shuffling approach. The starting geneswill be obtained from various natural isolates of HCV. In addition,related genes from other viruses can be used to increase the number ofdifferent recombinants that are generated. A library of related,chimeric variants of HCV antigens are then generated and this librarywill be screened for induction of widely crossreactive immune responses.The screening can be done directly in vivo by injecting individualvariants into test animals, such as mice or monkeys. Either purifiedrecombinant proteins or DNA vaccines encoding the relevant genes areinjected. Typically, a booster injection is given 2-3 weeks after thefirst injection. Thereafter, the sera of the test animals are collectedand these sera are tested for the presence of antibodies that reactagainst multiple HCV virus isolates.

Before the in vivo testing is initiated, the antigens can bepre-enriched in vitro for antigens that are recognized by polyclonalantisera derived from previously infected patients or test animals.Alternatively, monoclonal antibodies that are specific for variousstrains of HCV are used. The screening is performed using phage displayor ELISA assays. For example, the antigen variants are expressed onbacteriophage M13 and the phage are then incubated on plates coated withantisera derived from patients or test animals infected with various HCVisolates. The phage that bind to the antibodies are then eluted andfurther analyzed in test animals for induction of crossreactiveantibodies.

Example 11 Evolution of Chimeric Allergens that Induce Broad ImmuneResponses and Have Reduced Risk of Inducing Anaphylactic Reactions

Specific immunotherapy of allergy is performed by injecting increasingamounts of the given allergens into the patients. The therapy typicallyalters the types of allergen-specific immune responses from a dominatingT helper 2 (T_(H)2) type response to a dominating T helper 1 (T_(H)1)type response. However, because allergic patients have increased levelsof IgE antibodies specific for the allergens, the immunotherapy ofallergy involves a risk of IgE receptor mediated anaphylactic reactions.

T helper (T_(H)) cells are capable of producing a large number ofdifferent cytokines, and based on their cytokine synthesis pattern T_(H)cells are divided into two subsets (Paul and Seder (1994) Cell 76:241-251). T_(H)1 cells produce high levels of IL-2 and IFN-gamma and noor minimal levels of IL-4, IL-5 and IL-13. In contrast, T_(H)2 cellsproduce high levels of IL-4, IL-5 and IL-13, whereas IL-2 and IFN-gammaproduction is minimal or absent. T_(H)1 cells activate macrophages,dendritic cells and augment the cytolytic activity of CD8+ cytotoxic Tlymphocytes and NK cells (Id.), whereas T_(H)2 cells provide efficienthelp for B cells and they also mediate allergic responses due to thecapacity of T_(H)2 cells to induce IgE isotype switching anddifferentiation of B cells into IgE secreting cell (De Vries andPunnonen (1996) In Cytokine regulation of humoral immunity: basic andclinical aspects. Eds. Snapper, C. M., John Wiley & Sons, Ltd., WestSussex, UK, pp. 195-215

This Example describes methods to generate chimeric allergens that canbroadly modulate allergic immune responses. This can be achieved by DNAshuffling of related allergen genes to generate chimeric genes. Inaddition, chimeric/mutated allergens are less likely to be recognized bypreexisting IgE antibodies of the patients. Importantly, allergenvariants that are not recognized by IgE antibodies can be selected usingpatient sera and negative selection (FIG. 13).

As one example, chimeric allergen variants of Der p2, Der f), Tyr p2 Lepd2 and Gly d2 allergens are generated. These house dust mite allergensare very common in exacerbating allergic and asthmatic symptoms, andimproved means to downregulate such allergic immune responses aredesired. House dust mites can be used as sources of the genes. Thecorresponding genes are shuffled using family DNA shuffling and ashuffled library is generated. Phage display is used to excludeallergens that are recognized by antibodies from allergic individuals.It is particularly important is to exclude variants that are recognizedby IgE antibodies. Phage expressing the allergen variants are incubatedwith pools of sera derived from allergic individuals. The phage that arerecognized by IgE antibodies are removed, and the remaining allergensare further tested in vitro and in vivo for their capacity to activateallergen-specific human T cells (FIG. 14). Because immunotherapy ofallergy is believed to function through induction of a dominating T_(H)1response as compared to allergic T_(H)2 response, efficient T cellactivation and induction of a T_(H)1 type response by allergen variantsis used as a measure of the efficacy of the allergens to modulateallergic T cell responses.

The optimal allergen variants are then further tested in vivo bystudying skin responses after injections to the skin. A stronginflammatory response around the injection site is an indication ofefficient T cell activation, and the allergen variants that induce themost efficient delayed type T cell response (typically observed 24 hoursafter the injection) are the best candidates for further studies in vivoto identify allergens that effectively downregulate allergic immuneresponses. Accordingly, these allergen variants re analyzed for theircapacity to inhibit allergic responses in allergic, atopic and asthmaticindividuals. The screening of allergen variants is further illustratedin FIG. 13 and FIG. 14.

Example 12 Evolution of Cancer Antigens that Induce Efficient Anti-TumorImmune Responses

Several cancer cells express antigens that are present at significantlyhigher levels on the malignant cells than on other cells in the body.Such antigens provide excellent targets for preventive cancer vaccinesand immunotherapy of cancer. The immunogenicity of such antigens can beimproved by DNA shuffling. In addition, DNA shuffling provides means toimprove expression levels of cancer antigens.

This Example describes methods to generate cancer antigens that canefficiently induce anti-tumor immune responses by DNA shuffling ofrelated cancer antigen genes. Libraries of shuffled melanoma-associatedglycoprotein (gp100/pmel17) genes (Huang et al. (1998) J. Invest.Dermatol. 111: 662-7) are generated. The genes can be isolated frommelanoma cells obtained from various patients, who may have mutations ofthe gene, increasing the diversity in the starting genes. In addition, agp100 gene can be isolated from other mammalian species to furtherincrease the diversity of starting genes. A typical method for theisolation of the genes is RT-PCR. The corresponding genes are shuffledusing single gene DNA shuffling or family DNA shuffling and a shuffledlibrary is generated.

The shuffled gp100 variants, either pools or individual clones, aresubsequently injected into test animals, and the immune responses arestudied (FIG. 15). The shuffled antigens are either expressed in E. coliand recombinant, purified proteins are injected, or the antigen genesare used as components of DNA vaccines. The immune response can beanalyzed for example by measuring anti-gp100 antibodies, as previousstudies indicate that the antigen can induce specific antibody responses(Huang et al., supra.). Alternatively, the test animals that can bechallenged by malignant cells expressing gp100. Animals that have beenefficiently immunized will generate cytotoxic T cells specific for gp100and will survive the challenge, whereas in non-immunized or poorlyimmunized animals the malignant cells will efficiently grow eventuallyresulting in lethal expansion of the cells. Furthermore, antigens thatinduce cytotoxic T cells that have the capacity to kill cancer cells canbe identified by measuring the capacity of T cells derived fromimmunized animals to kill cancer cells in vitro. Typically the cancercells are first labeled with radioactive isotopes and the release ofradioactivity is an indication of tumor cell killing after incubation inthe presence of T cells from immunized animals. Such cytotoxicity assaysare known in the art.

The antigens that induce highest levels of specific antibodies and/orcan protect against the highest number of malignant cells can be chosenfor additional rounds of shuffling and screening. Mice are useful testanimals because large numbers of antigens can be studied. However,monkeys are a preferred test animal, because the MHC molecules ofmonkeys are very similar to those of humans.

To screen for antigens that have optimal capacity to activateantigen-specific T cells, peripheral blood mononuclear cells frompreviously infected or immunized humans individuals can be used. This isa particularly useful method, because the MHC molecules that willpresent the antigenic peptides are human MHC molecules. Shuffled cancerantigens that induce cytotoxic T cells that have the capacity to killcancer cells can be identified by measuring the capacity of T cellsderived from immunized animals to kill cancer cells in vitro. Typicallythe cancer cells are first labeled with radioactive isotopes and therelease of radioactivity is an indication of tumor cell killing afterincubation in the presence of T cells from immunized animals. Suchcytotoxicity assays are known in the art.

Example 13 Evolution of Autoantigens that Induce Efficient ImmuneResponses

Autoimmune diseases are characterized by an immune response directedagainst self antigens expressed by the host. Autoimmune responses aregenerally mediated by T_(H)1 cells that produce high levels of IL-2 andIFN-gamma. Vaccines that can direct autoantigen specific T cells towardsT_(H)2 phenotype producing increased levels of IL-4 and IL-5 would bebeneficial. For such vaccines to work, the vaccine antigens have to beable to efficiently activate specific T cells. DNA shuffling can be usedto generate antigens that have such properties. To optimally induceT_(H)2 cell differentiation it may be beneficial to coadministercytokines that have been shown to enhance T_(H)2 cell activation anddifferentiation, such as IL-4 (Racke et al. (1994) J. Exp. Med. 180:1961-66).

This Example describes methods for generating autoantigens that canefficiently induce immune responses. DNA shuffling is performed onrelated autoantigen genes. For example, libraries of shuffled myelinbasic proteins, or fragments thereof (Zamvil and Steinman (1990) Ann.Rev. Immunol. 8: 579-621); Brocke et al. (1996) Nature 379: 343-46) aregenerated. MBP is considered to be an important autoantigen in patientswith multiple sclerosis (MS). The genes encoding MBP from at leastbovine, mouse, rat, guinea pig and human have been isolated providing anexcellent starting point for family shuffling. A typical method for theisolation of the genes is RT-PCR. The shuffled MBP variants, eitherpools or individual clones, are subsequently injected into test animals,and the immune responses are studied. The shuffled antigens are eitherexpressed in E. coli and recombinant, purified proteins are injected, orthe antigen genes are used as components of DNA vaccines or viralvectors. The immune response can be analyzed for example by measuringanti-MBP antibodies by ELISA. Alternatively, the lymphocytes derivedfrom immunized test animals are activated with MBP, and the T cellproliferation or cytokine synthesis is studies. A sensitive assays forcytokine synthesis is ELISPOT (McCutcheon et al. (1997) J. Immunol.Methods 210: 149-66). Mice are useful test animals because large numbersof antigens can be studied. However, monkeys are a preferred testanimal, because the MHC molecules of monkeys are very similar to thoseof humans.

To screen for antigens that have optimal capacity to activate MBPspecific T cells peripheral blood mononuclear cells from patients withMS can also be used. This is a particularly useful method, because theMHC molecules that will present the antigenic peptides are human MHCmolecules. Shuffled antigens that activate MBP specific T cells can beidentified by measuring the capacity of T cells derived from MS patientsto proliferate or produce cytokines upon culture in the presence of theantigen variants. Such assays are known in the art. One such assay isELISPOT (McCutcheon et al., supra.). An indication of the efficacy of anMBP variant to activate specific T cells is also the degree of skininflammation when the antigen is injected into the skin of a patientwith MS. Strong inflammation is correlated with strong activation ofantigen-specific T cells. Improved activation of MBP specific T cells,particularly in the presence of IL-4, is likely to result in enhancedT_(H) ² cell responses, which are beneficial in the treatment of MSpatients.

Example 14 Method of Optimizing the Immunogenicity of Hepatitis BSurface Antigen

This Example describes methods by which the envelope protein sequence ofthe hepatitis B virus can be evolved to provide a more immunogenicsurface antigen. Such a protein is important for vaccination of lowresponders and for immunotherapy of chronic hepatitis B.

Background

Current HBV vaccines (Merck, SKB) are based on the immunogenicity of theviral envelope protein and contain the Major (or Small) form of theenvelope protein produced as particles in yeast. These particles induceantibodies to the major surface antigen (HBsAg) which can protectagainst infection when antibody levels are at least 10milli-International Units per milliliter (mU/ml). These recombinantprotein preparations are not capable of inducing humoral immunity inchronic carriers (some 300 million cases worldwide) the induction ofwhich would be important to control virus spread. Moreover, certainindividuals respond poorly to the vaccine (up to 30-50% of vaccinees insome groups) and do not develop protective levels of antibody. Theinclusion of the natural epitope sequences contained in the Middle orLarge forms of the viral envelope protein has been used as a method toincrease the immunogenicity of vaccine preparations. An alternativemethod is to introduce new (i.e., not present in the natural virussequence) helper T-cell epitopes into the HBsAg sequence using DNAshuffling technology.

Method

DNA sequences of HBsAg from different subtypes of HBV (e.g., ayw andadr) and the related woodchuck hepatitis virus are prepared forshuffling. Comparison of the genes encoding these proteins suggests thatrecombination would occur at least ten times within 850 base pairs whenshuffling the ayw and woodchuck hepatitis virus (WHV) DNA sequences.Nucleotide and amino acid sequences of portions of different subtypes ofHBV are shown in FIG. 17.

The sequence of the main HBsAg B-cell antigenic site (the “a” epitope)can be retained in the protein sequence by including the codingsequences of the external “a” loop in the final protein preparation.Peptide analogue(s) for the “a” epitope of HBsAg have been described(Neurath et al. (1984) J. Virol. Methods 9:341-346), and theimmunogenicity of the “a” epitope has been demonstrated (Bhatnagar etal. (1982) Proc. Nat'l. Acad. Sci. USA 79: 4400-4404). HBsAg and WHsAgshare the major “a” determinant, and chimps can be protected by bothantigens (Cote et al. (1986) J. Virol. 60: 895-901). Likewise, importantCTL epitopes can be included in the protein in a defined way.

One can also easily introduce B or T (helper or CTL) epitopes from otherantigens into the shuffled HBsAg sequence. This may focus the immuneresponse to certain epitopes, independent of other potentially dominantepitopes from the same protein. Furthermore, the availability of the “a”loop on the HBsAg may provide a region of the envelope protein intowhich other artificial antigens or mimotopes could be included.

In all cases where a novel HBV envelope sequence is prepared to includea specific epitope (from HBV, another pathogen or a tumor cell),shuffling of the surrounding sequences in the HBV envelope will serve tooptimize expression of the protein and help to ensure that the immuneresponse is directed to the desired epitope.

Several methods of analyzing and utilizing shuffled HBsAg sequences aredescribed below.

A. Modulating Expression Levels of HBsAg

Shuffled HBsAg sequences are introduced into cells in culture and theability to direct expression of secreted HBsAg (measured with clinicalkits for HBsAg expression) is evaluated. This can be used to identifyshuffled HBsAg sequences which exhibit optimized HBSAg expressionlevels. Such coding sequences are particularly interesting for DNAvaccination.

B. Circumventing Low Responsiveness to the HBsAg

Shuffled HBsAg sequences are evaluated for their ability to induce animmune response to the clinically relevant HBsAg epitopes. This can bedone using mice of the H-2s and H-2f haplotypes, which respond poorly ornot at all to HBsAg protein immunization. In these experiments, one canverify that antibodies are generated to the main “a” epitope in the Sprotein, and a second protective epitope in the PreS2 region (a linearsequence).

The PreS2 and S coding sequences for the envelope protein (HBsAg) fromthe HBV ayw subtype (plasmid pCAG-M-Kan; Whalen) and the WHV (plasmidpWHV8 from ATCC) are amplified from the two plasmids by PCR andshuffled. Examples of suitable primers for PCR amplification are shownin FIG. 18. The shuffled library of sequences is cloned into anHBsAg-expression vector and individual colonies are chosen forpreparation of plasmid DNA. The DNA is administered to the test animalsand vectors which induce the desired immune response are identified andrecovered.

C. Presentation of Natural HBsAg CTL Epitopes by Evolved HBsAg Proteins

This example describes methods of using the evolved HBsAg protein topresent natural HBsAg CTL epitopes. Shuffling is used to increaseoverall immunogenicity of the HBsAg protein, as discussed above.However, some of the evolved HBsAg sequences are replaced with class Ior class II epitope sequences from the natural HBsAg protein in order tostimulate immunoreactivity specifically to these natural viral epitopes.Alternatively, the natural viral epitopes can be added to the evolvedprotein without loss of immunogenicity of the evolved HBsAg.

D. Expression of Tumor-Derived CTL Epitopes by Evolved HBsAg Proteins

This example describes methods of using the evolved HBsAg protein isused to express tumor-derived CTL epitopes. The overall immunogenicityof the HBsAg protein is increased by shuffling. However, some of theevolved HBsAg sequences are replaced with class I or class II epitopesequences from tumor cells in order to stimulate immunoreactivityspecifically to these natural viral epitopes. Alternatively, the tumorcells epitopes can be added to the evolved protein without loss ofimmunogenicity of the evolved HBsAg.

E. Expression of Mimotope Sequences by the HBsAg

This example describes the use of an evolved HBsAg protein forexpression of mimotope sequences. Again, the evolved HBsAg protein isused to increase overall immunogenicity of the protein. However, some ofthe evolved HBsAg sequences are replaced with mimotope sequences tostimulate immunoreactivity specifically to the natural sequence whichcross reacts with the mimotope. Alternatively, the mimotope sequencescan be added to the evolved protein without loss of immunogenicity ofthe evolved HBsAg.

Example 15 Fusion Proteins of the HBsAg Polypeptide and HIV gp120Protein

This Example describes the preparation of fusion proteins (“chimeras”)formed from the HBsAg polypeptide and the extracellular fragment gp 120of the HIV envelope protein, and their use as vaccines.

Background

When used as a vaccine, recombinant monomeric gp120 has failed to induceantibodies that have strong neutralizing activity with primary isolatesof the HIV virus. It has been suggested that oligomeric forms of the HIVenvelope protein which expose certain regions of the tertiary structurewould be better able to elicit virus-neutralizing antibodies (Parrin etal. (1997) Immunol. Lett. 57: 105-112; VanCott et al. (1997) J. Virol.71: 4319-4330;

In this Example, DNA shuffling is applied to this problem, in order toobtain gp120 polypeptides which adopt conformations slightly differentfrom those of previous preparations of recombinant gp120. To allow theindividual gp120 molecules to interact as oligomers, a fusion isprepared between gp120 sequences (on the N-terminus of the fusion) andHBsAg sequences (on the C-terminal of the fusion).

The N-terminal peptide sequence of the S region of the HBsAg polypeptideis a transmembrane structure which is locked into the membrane of theendoplasmic reticulum. The actual N-terminus of the S region as well asthe preS2 sequences are located in the lumenal part of the ER. They arefound on the outside of the final HBsAg particles. By placing the gp120sequences on the N-terminus of the HBsAg preS2 or S sequences, the gp120sequences are also located on the outside of the particles. The gp120molecules can thus be brought together in three-dimensional space tointeract as in the virus.

Since the exact conformation of the final chimera which will have themost appropriate immunogenicity cannot be predicted, DNA shuffling isemployed. The sequences of the HBsAg polypeptide, which functions as ascaffold, and of gp120 are both shuffled. Screening of the shuffledproducts can be performed by ELISA assay using antibodies (polyclonal ormonoclonal) which have previously been determined to have virusneutralizing activity.

Method

The sequences encoding the gp120 fragment of the HIV envelope proteinare preferably prepared as a synthetic gene to include codons which areoptimal for gene expression in mammals. The gp120 sequence willtypically include a signal sequence on its N-terminal end.

The gp120 sequences are inserted into the preS2 region of anHBsAg-expressing plasmid. In the preS2 region of the plasmid pMKan andits derivatives, an EcoRI site and an KhoI site are available forcloning. The gp120 sequences can be inserted between these two sites,which brings the gp120 closer to the start of the S coding sequences, orinto the EcoRI site alone, which leaves a spacer sequence of about 50amino acids between the gp120 sequence and the start of the S region ofthe HBsAg. These two different cloning strategies will give rise tochimeric molecules in which the gp120 sequences are located at differentdistances from the transmembrane region of the HBsAg sequence. This maybe advantageous in allowing the gp120 sequences to adopt conformationswhich are more suitable immunogens than monomeric gp120.

DNA shuffling of the entire chimeric sequence is carried out. Familyshuffling is preferred; this involves the preparation of severalgp120-HBsAg fusion proteins in which different gp120 and HBsAg (or WHV)sequences are used. An alignment of HBsAg nucleotide sequences is shownin FIG. 19. After shuffling of the different sequences, the products arecloned into an expression vector such as pMKan. Pools of clones from thelibrary of shuffled products are transfected into cultured cells and thesecretion of chimeric proteins is assayed with broadly reactiveantibodies to gp120. Positive clones can be further evaluated withparticular antibodies that have demonstrated HIV neutralizing activity,for example the anti-CD4 binding domain recombinant human monoclonalantibody, IgG1b12 (Kessler et al. (1997) AIDS Res. Hum. Retroviruses. 1:13: 575-582; Roben et al. (1994) J. Virol. 68: 4821-4828). Candidateclones can then be used to immunize mice and the antiserum obtained isevaluated for HIV virus-neutralizing activity in in vitro assays.

Because the gp120 molecule (approx. 1100 amino acids) is larger in sizethan the monomeric HBsAg preS2+S protein (282 amino acids), it is likelythat not every HBsAg monomer in an aggregated particle will contain agp120 sequence. Internal initiation of protein synthesis can take placeon the HBsAg coding sequences at the initiator methionine that marks thebeginning of the S region. Thus, the chimeric molecule (which containsthe gp120 sequences) will be mixed in the cell with the S region and themultimeric particles should assemble with an appropriate number ofchimeric polypeptides and native HBsAg S monomers. Alternatively, anS-expressing plasmid can be mixed with the plasmid expressing thechimera, or a single plasmid which expresses the chimera and the S formcan be constructed. A diagram of the resulting particles is shown inFIG. 20.

Example 16 DNA Shuffling of HSV-1 And HSV-2 Glycoproteins B and/or D asMeans to Induce Enhanced Protective Immune Responses

This Example describes the use of DNA shuffling to obtain HSVglycoprotein B (gB) and glycoprotein D (gD) polypeptides that exhibitimproved ability to induce protective immune responses uponadministration to a mammal. Epidemiological studies have shown thatprior infections with HSV-1 give partial protection against infectionswith HSV-2, indicating existence of cross-reactive immune responses.Based on previous vaccination studies, the main immunogenicglycoproteins in HSV appear to be gB and gD, which are encoded by 2.7 kband 1.2 kb genes, respectively. The gB and gD genes of HSV-1 are about85% identical to the corresponding gene of HSV-2, and the gB genes ofeach share little sequence identity with the gD genes. Baboon HSV-2 gBis appr. 75% identical to human HSV-1 or -2 gB, with rather longstretches of almost 90% identity. In addition, 60-75% identity is foundin portions of the genes of equine and bovine herpesviruses.

Family shuffling is employed using as substrates nucleic acids thatencode gB and/or gD from HSV-1 and HSV-2. Preferably, homologous genesare obtained from HSVs of various strains. An alignment of gD nucleotidesequences from HSV-1 and two strains of HSV-2 is shown in FIG. 7.Antigens encoded by the shuffled nucleic acids are expressed andanalyzed in vivo. For example, one can screen for improved induction ofneutralizing antibodies and/or CTL responses against HSV-1/HSV-2. Onecan also detect protective immunity by challenging mice or guinea pigswith the viruses. Screening can be done using pools or individualsclones.

Example 17 Evolution of HIV Gp120 Proteins for Induction of BroadSpectrum Neutralizing Ab Responses

This Example describes the use of DNA shuffling to generate immunogensthat crossreact among different strains of viruses, unlike the wild-typeimmunogens. Shuffling two kinds of envelope sequences can generateimmunogens that induce neutralizing antibodies against a third strain.

Antibody-mediated neutralization of HIV-1 is strictly type-specific.Although neutralizing activity broadens in infected individuals overtime, induction of such antibodies by vaccination has been shown to beextremely difficult. Antibody-mediated protection from HIV-1 infectionin vivo correlates with antibody-mediated neutralization of virus invitro.

FIG. 8 illustrates the generation of libraries of shuffled gp120 genes.gp120 genes derived from HIV-1DH12 and HIV-1IIIB(NL43) are shuffled. Thechimeric/mutant gp120 genes are then analyzed for their capacity toinduce antibodies that have broad spectrum capacity to neutralizedifferent strains of HIV. Individual shuffled gp120 genes areincorporated into genetic vaccine vectors, which are then introduced tomice by injection or topical application onto the skin. These antigenscan also be delivered as purified recombinant proteins. The immuneresponses are measured by analyzing the capacity of the mouse sera toneutralize HIV growth in vitro. Neutralization assays are performedagainst HIV-1DH12, HIV-1IIIB and HIV-189.6. The chimeras/mutants thatdemonstrate broad spectrum neutralization are chosen for further roundsof shuffling and selection. Additional studies are performed in monkeysto illustrate the capacity of the shuffled gp120 genes to provideprotection for subsequent infection with immunodeficiency virus.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference for allpurposes.

1. An recombinant multivalent antigenic polypeptide that comprises afirst antigenic determinant of a first polypeptide and at least a secondantigenic determinant from a second polypeptide. 2-53. (canceled)